Biomarkers for liver fibrosis

ABSTRACT

Methods and systems for diagnosing or prognosing liver fibrosis in a subject are provided. In some examples, such methods and systems can include detecting liver fibrosis-related molecules in a sample obtained from the subject, comparing expression of the molecules in the sample to controls representing expression values expected in a subject who does not have liver fibrosis or who has non-progressing fibrosis, and diagnosing or prognosing liver fibrosis in the subject when differential expression of the molecules between the sample and the controls is detected. Kits for the diagnosis or prognosis of liver fibrosis in a subject are also provided which include reagents for detecting liver fibrosis related molecules.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application No.61/785,225 filed Mar. 14, 2013, herein incorporated by reference in itsentirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

This invention was made with government support under 5P41RR018522-10awarded by the National Institutes of Health's National Center forResearch Resources, under 8 P41 GM103493-10 awarded by the NationalInstitute of General Medical Sciences, under R21-CA12619-01 andU24-CA-160019-01 awarded by the National Cancer Institute, andDE-AC05-76RLO01830 awarded by the Department of Energy. The Governmenthas certain rights in the invention.

FIELD

The present disclosure generally relates to methods and systems for thescreening and detection of persons having or at risk for liver fibrosis,as well as kits that can be used with such methods and systems.

BACKGROUND

To date pre-clinical and clinical applications of mass spectrometry(MS)-based proteomic techniques analyzing complex biofluids have fallenshort of expectations, largely due to deficiencies in both analyticalsensitivity and throughput. These deficiencies result in measurementstypically failing to confidently detect and quantify proteins atmoderate to low concentrations, or not providing sufficient sampleanalysis throughput for statistical relevance. Higher sensitivitytargeted MS analyses are currently utilized to address theseshortcomings [1, 2]; however, these often only analyze a small list ofproteins identified as biologically significant. While targeted MSmeasurements are increasingly common in clinical applications [3, 4],the limited number of proteins they examine does not necessarily reflectthe biodiversity across a population, making broad untargetedmeasurements useful in developing individual disease metrics fordiagnosis [5]. As the future of medicine proceeds toward a personalprofiling approach [6, 7], the potential for robust high throughputclinical measurements based upon MS is highly attractive if itsdeficiencies can be addressed.

An initial step in attaining broad untargeted measurements thatincreasingly retain the benefits of targeted analyses exploitstechnological advances such as faster separations, more effective ionsources, detectors with greater dynamic range, and MS measurements withboth higher resolution and accuracy. Advanced liquid-phase separationshave already been employed to provide a significant sensitivity increaseas illustrated by the higher number of proteins detected in liquidchromatography (LC)-MS-based studies [8]; however, the long LCseparations most compatible with blood samples are extremelytime-consuming. Fast gas-phase ion mobility spectrometry (IMS)separations that take place on the time scale of tens of millisecondsoffer an additional separation stage and a way of reducing the need forextended LC separation times. In an IMS separation, ions subject to anelectric field while traveling through a buffer gas separate quicklybased on ion shape, e.g. compact species drift faster than those withextended structures [9, 10]. IMS can be coupled between LC andorthogonal acceleration time-of-flight (TOF) MS stages, and by combiningthe three orthogonal separations into a single LC-IMS-MS instrumentationplatform, multidimensional high-resolution nested spectra are producedcontaining elution times, mass-to-charge ratios (m/z) and IMS drifttimes for all detectable ions in a sample [11, 12]

Liver fibrosis may result from a wide variety of conditions includingchronic alcohol exposure, hepatitis B virus (HBV) infection,non-alcoholic fatty liver disease (NAFLD), hepatitis C virus (HCV)infection, Wilson's disease, alpha-1-antitrypsin deficiency,hemochromatosis, primary biliary cirrhosis, primary sclerosingcholangitis, and autoimmune hepatitis. Chronic HCV is the leadingcontributor to chronic liver disease and represents a worldwide publichealth concern affecting an estimated 130-170 million people [16]. Theliver damage ensuing from HCV infection is also the leading cause ofliver transplants in the United States and Europe and a major burden onhealthcare services [17, 18]. In this disease, the liver elicits apersistent inflammatory and repair response known as fibrosis, which ischaracterized by the formation of fibrous tissue and scarring on theliver. Because the prognosis of HCV patients is related to thedevelopment of fibrosis and the risk of cirrhosis and hepatocellularcarcinoma, an accurate evaluation of fibrogenic progression is importantfor patient care.

Currently, liver biopsies are the primary technique for generatinginformation on the degree of fibrosis; however, they have multipledisadvantages, including risk of complications (e.g., major bleeding orinadvertent puncture of the lung, kidney, or colon), cost andoccasionally inaccurate findings due to small specimen size andvariability in histology evaluation. These disadvantages have spurredthe development of noninvasive methods that can reliably predict,diagnose and assess the degree of fibrosis [19, 20].

SUMMARY

The present disclosure provides methods of diagnosing or prognosingliver fibrosis in a subject. Such methods can include detecting ormeasuring expression of at least two (e.g., at least 3, at least 4, atleast 5 or at least 10) liver fibrosis-related molecules (such asproteins or nucleic acids) in a sample obtained from the subject,comparing the detected expression in the sample to controls representingexpression of the at least two (e.g., at least 3, at least 4, at least 5or at least 10) liver fibrosis-related molecules expected in a subjectwho does not have liver fibrosis or who has non-progressing liverfibrosis, and diagnosing or prognosing liver fibrosis in the subjectwhen there is differential expression of the at least two (e.g., atleast 3, at least 4, at least 5 or at least 10) liver fibrosis-relatedmolecules between the sample and the controls, such as an increase ordecrease in expression of at least 10% or at least 1.1 fold.

Also provided are one or more non-transitory computer-readable mediathat include computer-executable instructions causing a computing systemto perform the methods provided herein.

Systems for analyzing a sample (such as a sample obtained from a subjectsuspected of having liver fibrosis) obtained from a subject are alsoprovided. Such systems can include a means for measuring a level of atleast two (e.g., at least 3, at least 4, at least 5 or at least 10)liver fibrosis-related molecules the sample (such as an antibody, probe,or primer) and means for diagnosing or prognosing liver fibrosis in thesubject based on the level of the at least two liver fibrosis-relatedmolecules. In some examples, the system includes implemented rules fordetermining diagnosing or prognosing liver fibrosis (e.g., positive ornegative) based on the measured level of the at least two liverfibrosis-related molecules. In some examples, the system includesimplemented rules for comparing the measured level of the at least twoliver fibrosis-related molecules to a reference value, such as the levelof the at least two liver fibrosis-related molecules positive ornegative control (such as values expected in a subject who does not haveliver fibrosis or who has non-progressing liver fibrosis). In someexamples, the reference values are stored values. In some examples, thereference values are a level of the at least two liver fibrosis-relatedmolecules measured from a control sample by said means for measuring.The system can also include one or more means for implementing therules, whereby an indication of diagnosing or prognosing liver fibrosis(e.g., positive or negative) is provided based on the differentialexpression determined for the at least two liver fibrosis-relatedmolecules measured in the patient sample and the control (e.g.,reference value). Kits are also provided for the diagnosis or prognosisof liver fibrosis in a subject. Such kits can include reagents fordetecting at least two (e.g., at least 3, at least 4, at least 5 or atleast 10) liver fibrosis-related molecules (such as proteins or nucleicacids). In some embodiments, the kit has antibodies specific for atleast two (e.g., at least 3, at least 4, at least 5 or at least 10)different liver fibrosis-related proteins. In some embodiments, the kitfurther includes labeled secondary antibodies which can bind to theantibodies specific for the proteins liver fibrosis-related proteins. Insome embodiments, the kit has oligonucleotide probes or primers specificfor at least two (e.g., at least 3, at least 4, at least 5 or at least10) different liver fibrosis-related nucleic acid molecules.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1A is a schematic representation of the ESI-IMS-MS regions of theoverall platform. The smaller conformational ions (green circles) andlarger conformational ions (red circles) illustrate the size separationin the IMS drift cell.

FIG. 1B provides a multiplexed nested IMS spectrum on the left showsboth IMS drift time (x-axis) and m/z (y-axis) for a 9 peptide mixture.In this spectrum, 8 ion packets were released into the drift cellsimultaneously. The corresponding de-multiplexed spectrum with all ionsdeconvoluted to their correct drift times is shown on the right. Theintensity in the nested spectra is represented by color, with red beingthe most intense and blue being the least.

FIG. 1C shows the results of adding LC to LC-IMS-MS analyses. The plotsillustrate the LC dimension total ion chromatogram (x-axis) and IMSdrift time (y-axis) for a human serum dataset before (left) and after(right) de-multiplexing. The intensity in the nested spectra isrepresented by color, with red being the most intense and blue being theleast.

FIG. 2A shows MS spectra for the LTQ Orbitrap Velos (left) and IMS-MS(right). The plots illustrate the reduction in noise in the IMS-MSspectrum and demonstrate how additional drift time information greatlysimplifies peak detection, ultimately leading to more identifications.

FIG. 2B shows an LC-IMS-MS spectra with (Dynorphin A porcine)³⁺(circled) spiked at 100 pg/mL into human serum. The IMS-MS spectra aredisplayed as two-dimensional nested plots for IMS drift time (x-axis)and m/z (y-axis). The intensity in the nested spectra is represented bycolor, with red being the most intense and blue being the least.

FIG. 3 illustrates the categorization of 60 HCV patients following livertransplant. Blood samples from the 60 HCV patients following livertransplant were utilized. Based on the amount of fibrosis occurring ineach patient's liver several months to a few years following theprocedure, the patients were categorized into non-, slow or fastprogressing liver fibrosis groups. A biostatistician selected patientsfor the study so that a non-progressor patient could be donor, age andcold ischemia time matched to either a slow or fast progressor,resulting in 30 well-annotated patient pairs.

FIG. 4A provides heat maps illustrating the relative log 2 intensitychange for proteins with significant differential expression showing upin at least 21 datasets by peptides when non-progressors are compared toeither slow progressors (left) or fast progressors (right). Each columnrepresents one of the 60 patients and each row one of the 136 proteinsshown in Table 1. Trends between the patient groups are clearlyobserved, but biodiversity in the human population is also discerned.

FIG. 4B provides heat maps illustrating the categorization of selectedproteins with significant differential expression into four groups basedon correlation with liver metabolism, innate immune response, oxidativestress or liver fibrosis. Gene names given in UniProtKB are used asabbreviated protein names. The twenty-five selected proteins are shownto illustrate the trend of each category. Decrease in relativeexpression is shown by green and increase in relative expression isshown by red (missing values are shown as grey).

FIG. 5A illustrates the categorization of 60 HCV infected non-transplantpatients into two categories, Stages 0 & 1 and Stages 4, 5 & 6. Bloodsamples from the 60 HCV infected non-transplant patients were utilizedfor comparison and validation of the transplant patient results.

FIG. 5B provides heat maps illustrating the relative log 2 intensitychange for proteins showing significant differential expression in thenon-transplant patient groups. Each column represents one of the 60patients and each row one of the 78 significant proteins shown in Table5. Trends between the patient groups are clearly observed, butbiodiversity in the human population is also discerned. Decrease inrelative abundance is shown by green and increase by red.

FIG. 6 illustrates Western blot (left) and LC-IMS-MS abundance (right)analyses of certain liver fibrosis patients where fast progressors (FP)were analyzed against their matched controls (NP). Patient fibrosisstage is noted on the bar graphs for the LC-IMS-MS abundance data.Patient pair is abbreviated as PP for clarity. For the Western blots anequal amount (5 μg) of each serum protein sample was loaded. Targetswere proteins involved in liver metabolism (F2: patient pairs 22 and16), the innate immune system (C4A: patient pairs 21 and 29 (the Westerntargeted C4 β-chain, a cleavage product of C4A), oxidative stress(QSOX1: patient pairs 24 and 22) and liver fibrosis (ECM1: patient pairs21 and 23 and LGALS3BP: patient pairs 24 and 21).

FIG. 7 is a schematic drawing showing the statistical processingpipeline for the verification non-transplant generated data.

FIG. 8 is a scores plot of the raw peptide abundance values. A sppPCAanalysis results in four distinct clusters of data based upon samplepreparation and instrument analysis batching. Clusters 4 and 2 were usedfor further statistical analysis.

FIGS. 9A-9D show quality control processing diagnostic figures. (A), (C)The correlation coefficient of log₁₀ peptide abundance values acrossLC-MS analyses. (B), (D) The statistical analysis of the log₂ robustMahalanobis distances (rMd) to determine if an LC-MS run should beremoved from the dataset. The red horizontal line represents a criticalvalue associated with a significance level of 0.0001. Blue downwardtriangles represent single technical replicates and red triangles, ifpresent, are for replicates where all associated technical replicatesare above the line.

FIG. 10 is a plot showing the principal component analysis of the finalprotein candidate list. The first principal component explains 29% ofthe total variation in the protein abundance data, and discriminates thefibrosis categories.

SEQUENCE LISTING

SEQ ID NOS: 1-5544 show peptide sequences that were detected using thedisclosed methods.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Abbreviations and Terms

The following explanations of terms and methods are provided to betterdescribe the present disclosure and to guide those of ordinary skill inthe art in the practice of the present disclosure. The singular forms“a,” “an,” and “the” refer to one or more than one, unless the contextclearly dictates otherwise. For example, the term “comprising a protein”includes single or plural proteins and is considered equivalent to thephrase “comprising at least one protein.” The term “or” refers to asingle element of stated alternative elements or a combination of two ormore elements, unless the context clearly indicates otherwise. As usedherein, “comprises” means “includes.” Thus, “comprising A or B,” means“including A, B, or A and B,” without excluding additional elements.

Unless explained otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood to one of ordinaryskill in the art to which this disclosure belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present disclosure, suitable methods andmaterials are described below. The materials, methods, and examples areillustrative only and not intended to be limiting. While the methodshave been described and were utilized in testing, it is to be distinctlyunderstood that the disclosure is not limited to any particular form oftesting utilized, but is intended to include all methods that arecapable of detecting the materials that are set forth in the claims. Allreferences provided herein are incorporated by reference, as are thesequences associated with the Gen Bank Accession number provided herein(sequences available on Mar. 14, 2013).

Alarm: A notification (such as an audio or visual (e.g., color, picture,or text) notification) indicating the presence or absence of liverfibrosis, the presence or absence of fast-progressing liver fibrosis,the presence or absence of slow-progressing liver fibrosis, and/or thepresence or absence of non-progressing liver fibrosis. The alarm may beactivated when analysis of a subject's sample yields results meetingcriteria for liver fibrosis, fast-progressing liver fibrosis,slow-progressing liver fibrosis, and/or non-progressing liver fibrosis.The criteria may be determined by the manufacturer of a kit, system, ordevice that includes the alarm or may, alternatively, be activated basedupon a standard as entered by a clinician or other end-user.

Antibody: A polypeptide ligand including at least a light chain or heavychain immunoglobulin variable region which specifically recognizes andbinds an epitope of an antigen, such as a liver fibrosis marker or afragment thereof (such as the proteins or peptides listed in any ofTables 1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544).Antibodies are composed of a heavy and a light chain, each of which hasa variable region, termed the variable heavy (V_(H)) region and thevariable light (V_(L)) region. Together, the V_(H) region and the V_(L)region are responsible for binding the antigen recognized by theantibody. In one example, an antibody specifically binds to one of theproteins or peptides listed in any of Tables 1, 2, 3, 5, 6 and 12, aswell as any of SEQ ID NOS: 1-5544, but not other proteins (such as otherproteins found in human serum or plasma).

This includes intact immunoglobulins and the variants and portions ofthem well known in the art, such as Fab′ fragments, F(ab)′₂ fragments,single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (for example, humanized murineantibodies), heteroconjugate antibodies (such as, bispecificantibodies). See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Ku by, J., Immunology, 3^(rd) Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda and kappa. There are five main heavy chainclasses (or isotypes) which determine the functional activity of anantibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991). The Kabat database is now maintainedonline. The sequences of the framework regions of different light orheavy chains are relatively conserved within a species. The frameworkregion of an antibody, that is the combined framework regions of theconstituent light and heavy chains, serves to position and align theCDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of anantigen. The CDRs of each chain are typically referred to as CDR1, CDR2,and CDR3, numbered sequentially starting from the N-terminus, and arealso typically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds to a particular liverfibrosis marker will have a specific V_(H) region and the V_(L) regionsequence, and thus specific CDR sequences. Antibodies with differentspecificities (i.e. different combining sites for different antigens)have different CDRs. Although it is the CDRs that vary from antibody toantibody, only a limited number of amino acid positions within the CDRsare directly involved in antigen binding. These positions within theCDRs are called specificity determining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected. Monoclonal antibodies areproduced by methods known to those of skill in the art, for instance bymaking hybrid antibody-forming cells from a fusion of myeloma cells withimmune spleen cells. Monoclonal antibodies include humanized monoclonalantibodies.

A “polyclonal antibody” is an antibody that is derived from differentB-cell lines. Polyclonal antibodies are a mixture of immunoglobulinmolecules secreted against a specific antigen, each recognizing adifferent epitope. These antibodies are produced by methods known tothose of skill in the art, for instance, by injection of an antigen intoa suitable mammal (such as a mouse, rabbit or goat) that induces theB-lymphocytes to produce IgG immunoglobulins specific for the antigenwhich are then purified from the mammal's serum.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds to one of theproteins or peptides listed in any of Tables 1, 2, 3, 5, 6 and 12, aswell as any of SEQ ID NOS: 1-5544.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they are substantially identical to human immunoglobulin constantregions, e.g., at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. Humanized immunoglobulins can beconstructed by means of genetic engineering (see for example, U.S. Pat.No. 5,585,089).

Array: An arrangement of molecules, such as biological macromolecules(such as peptides, antibodies, aptamers, aptazymes or nucleic acidmolecules, such as nucleic acid probes), in addressable locations on orin a substrate. A “microarray” is an array that is miniaturized so as torequire or be aided by microscopic examination for evaluation oranalysis.

The array of molecules (“features”) makes it possible to carry out avery large number of analyses on a sample at one time. In certainexample arrays, one or more molecules (such as oligonucleotide probes,aptamers, aptazymes, or antibodies) will occur on the array a pluralityof times (such as twice), for instance to provide internal controls. Thenumber of addressable locations on the array can vary, for example fromat least two, at least 3, at least 4, at least 5, at least 10, at least20, at least 30, at least 50, at least 75, at least 100, at least 150,at least 200, at least 300, at least 500, least 550, at least 600, atleast 800, at least 1000, at least 10,000, or more. In particularexamples, an array includes nucleic acid molecules, such asoligonucleotide sequences that are at least 15 nucleotides in length,such as about 15-40 nucleotides in length. In particular examples, anarray includes aptamers, aptazymes, or antibodies to detect targetproteins.

In particular examples, an array includes oligonucleotide probes orprimers which can be used to detect liver fibrosis-associated nucleicacids. Protein-based arrays include probe molecules that are or includeproteins, or where the target molecules are or include proteins, andarrays including nucleic acids to which proteins are bound, or viceversa. For example, such arrays can be used to detect any combination ofat least two of the liver fibrosis-related proteins (or nucleic acidsencoding such proteins) listed in any of Tables 1, 2, 3, 5, 6 and 12, aswell as any of SEQ ID NOS: 1-5544, such as at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 15, at least 20, at least 22, at least 25, at least 30, at least35, at least 40, or at least 45 of the molecules listed in any of Tables1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544.

Within an array, each arrayed sample is addressable, in that itslocation can be reliably and consistently determined within at least twodimensions of the array. The feature application location on an arraycan assume different shapes. For example, the array can be regular (suchas arranged in uniform rows and columns) or irregular. Thus, in orderedarrays the location of each sample is assigned to the sample at the timewhen it is applied to the array, and a key may be provided in order tocorrelate each location with the appropriate target or feature position.Often, ordered arrays are arranged in a symmetrical grid pattern, butsamples could be arranged in other patterns (such as in radiallydistributed lines, spiral lines, or ordered clusters). Addressablearrays usually are computer readable, in that a computer can beprogrammed to correlate a particular address on the array withinformation about the sample at that position (such as hybridization orbinding data, including for instance signal intensity). In some examplesof computer readable formats, the individual features in the array arearranged regularly, for instance in a Cartesian grid pattern, which canbe correlated to address information by a computer.

Binding affinity: Affinity of one molecule for another, such as anantibody, aptamer, or aptazyme for an antigen (for example, the proteinsor peptides shown in any of Tables 1, 2, 3, 5, 6 and 12, as well as anyof SEQ ID NOS: 1-5544). In one example, affinity is calculated by amodification of the Scatchard method described by Frankel et al., Mol.Immunol., 16:101-106, 1979. In another example, binding affinity ismeasured by an antigen/antibody dissociation rate. In yet anotherexample, a high binding affinity is measured by a competitionradioimmunoassay. In several examples, a high binding affinity is atleast about 1×10⁻⁸ M. In other examples, a high binding affinity is atleast about 1.5×10⁻⁸, at least about 2.0×10⁻⁸, at least about 2.5×10⁻⁸,at least about 3.0×10⁻⁸, at least about 3.5×10⁻⁸, at least about4.0×10⁻⁸, at least about 4.5×10⁻⁸, or at least about 5.0×10⁻⁸ M. Thedetermination that a particular agent binds substantially only to asingle liver fibrosis marker peptide may readily be made by using oradapting routine procedures. One suitable in vitro assay makes use ofthe Western blotting procedure (described in many standard texts,including Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL,New York, 1999).

Control: A sample, standard, or reference value(s) used for comparisonwith a test sample. In some embodiments, the control is a sampleobtained from a healthy patient (or plurality of patients) (alsoreferred to herein as a “normal” control), such as a normal sample(e.g., one from a patient(s) that does not have liver fibrosis). In someembodiments, the control is a historical control or standard value(s)(e.g., a previously tested control sample or group of samples thatrepresent baseline or normal values). In some embodiments the control isa standard value representing the average value (or average range ofvalues) obtained from a plurality of patient samples, such as an averagevalue of the amount (e.g., relative or absolute) for each protein orpeptide listed in any of Tables 1, 2, 3, 5, 6 and 12, as well as any ofSEQ ID NOS: 1-5544, in patients without liver fibrosis or in patientswith non-progressing liver fibrosis.

A control can also be represented by a reference value or range ofvalues representing an amount of activity or expression determined to berepresentative of a given condition. Reference values can include arange of values, real or relative expected to occur under certainconditions. These values can be compared with experimental values todetermine if a given molecule is up-regulated or down-regulated in aparticular sample. In one example, a reference value or range of valuesrepresents an amount of activity or expression of a liver fibrosisrelated protein or peptide in a sample, such as a sample from a patientthat does not have liver fibrosis (and who may or may not have a livertransplant) or from a subject with no-progressing liver fibrosis. Thisvalue can then be used to determine if the subject from whom a testsample was obtained has liver fibrosis (such as fast- orslow-progressing liver fibrosis) or is at high risk for developing liverfibrosis (such as fast- or slow-progressing liver fibrosis) by comparingthis reference value of expression to the level of expression detectedin the test sample. In a particular example, a significant change inexpression or activity in four or more liver fibrosis related molecules(such an increase or decrease of at least 10%, at least 20%, at least30%, at least 40%, at least 50%, at least 75%, at least 80%, at least90%, at least 100%, at least 150%, at least 200%, or at least 300% forthose in any of Tables 1, 2, 3, 5, 6 and 12, as well as any of SEQ IDNOS: 1-5544) in a test sample as compared to such a reference valueindicates that the subject has liver fibrosis or is at a high risk forliver fibrosis. In a particular example, a significant change inexpression or activity in four or more liver fibrosis related molecules(such an increase or decrease of at least 1.2 fold, at least 1.3 fold,at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least2.1 fold, at least 2.2 fold, or at least 2.5 fold for those in any ofTables 1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544) in atest sample as compared to such a reference value indicates that thesubject has liver fibrosis or is at a high risk for liver fibrosis.

Detect: To measure, determine, or identify the existence, occurrence,presence, or fact of something. General methods of detecting are knownto the person of ordinary skill in the art and may be supplemented withthe protocols and reagents disclosed herein. For example, includedherein are methods of detecting a protein or peptide shown in any ofTables 1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544.Detection can be qualitative or quantitative and may be direct orindirect. Detection can be in bulk, so that a macroscopic number ofmolecules can be observed simultaneously. Detection can also includeidentification of signals from single molecules using microscopy andsuch techniques as total internal reflection to reduce background noise.

Diagnosis: The process of identifying a disease by its signs, symptomsand/or results of one or more tests, such as those provided herein. Theconclusion reached through that process is also called “a diagnosis.”Forms of testing commonly performed include blood tests, medicalimaging, genetic analysis, urinalysis, biopsy and analysis of biologicalsamples obtained from a subject. In one example diagnosis is determiningwhether a subject has liver fibrosis. In another example, diagnosis isdetermining whether a subject has fast progressing liver fibrosis. Inyet another example, diagnosis is determining whether a subject has slowprogressing liver fibrosis.

Differential expression: A difference, such as an increase or decrease,in the conversion of the information encoded in a gene (such as a liverfibrosis related gene) into messenger RNA, the conversion of mRNA to aprotein, or both. In an example, the difference is relative to a controlor reference value, such as an amount of protein expression that isexpected in a subject who does not have liver fibrosis. In anotherexample, the difference is relative to a control or reference value,such as an amount of protein or gene expression that is expected in asubject that has a non-progressing liver fibrosis. In another example,the difference is relative to a control or reference value, such as anamount of protein or gene expression that is expected in a subject thathas a slow-progressing liver fibrosis. Detecting differential expressioncan include measuring a change in gene or protein expression, such as achange in amount (e.g., qualitative or quantitative) of one or moreliver fibrosis related proteins, such as those listed in any of Tables1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544.

Downregulated or inactivated: When used in reference to the expressionof a protein, refers to any process which results in a decrease inproduction or expression of a protein, such as a decrease of at least10%, at least 20%, at least 50%, or at least 90%, such as an at least1.2-fold, at least 1.5 fold, at least 2-fold, at least 3-fold, at least4-fold, or at least 5-fold decrease. Protein downregulation includes anysignificant decrease in the detectable protein in a sample. In certainexamples, the detectable protein in a sample decreases by at least 10%,at least 20%, at least 50%, or at least 90%, such as at least 1.2-fold,at least 1.5 fold, at least 2-fold, at least 3-fold or at least 4-fold,as compared to a control (such an amount of the detectable protein in asample from a normal subject, such as one without liver fibrosis). Inone example, a control is a relative amount of protein expression in aserum sample from a subject who does not have liver fibrosis. Similarly,when used in reference to the expression of a nucleic acid, such as cDNAor mRNA, refers to any process which results in a decrease in productionor expression of a nucleic acid, such as a decrease of at least 10%, atleast 20%, at least 50%, or at least 90%, such as an at least 1.2-fold,at least 1.5 fold, at least 2-fold, at least 3-fold, at least 4-fold, orat least 5-fold decrease.

Label: A detectable compound. In some examples, a label is conjugateddirectly or indirectly to another molecule, such as an antibody,protein, or nucleic acid probe, to facilitate detection of thatmolecule. In this case, the molecule such as an antibody, nucleic acidprobe, or protein is labeled with the detectable compound. The label canbe capable of detection by, for example, ELISA, spectrophotometry, flowcytometry, or microscopy. Specific, non-limiting examples of labelsinclude fluorophores, chemiluminescent agents, enzymatic linkages, andradioactive isotopes. Methods for labeling and guidance in the choice oflabels appropriate for various purposes are discussed for example inSambrook et al. (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor, N.Y., 1989) and Ausubel et al. (In Current Protocols inMolecular Biology, John Wiley & Sons, New York, 1998). In a particularexample, a label is conjugated to an antibody or aptamer specific for aprotein or peptide disclosed in any of Tables 1, 2, 3, 5, 6 and 12, aswell as any of SEQ ID NOS: 1-5544 to allow for the diagnosis of liverfibrosis. In another example, a label is conjugated to a secondaryantibody specific for an antibody which is in turn specific for aprotein or peptide disclosed in any of Tables 1, 2, 3, 5, 6 and 12, aswell as any of SEQ ID NOS: 1-5544 to allow for the diagnosis of liverfibrosis.

Liver Fibrosis: The scarring process that occurs in the liver inresponse to injury to the liver and involves excessive accumulation ofextracellular matrix proteins such as collagen. Liver fibrosis is oftena result of chronic inflammation of the liver due to, for example,infection with hepatitis C virus (HCV). Chronic inflammation leads tochanges in liver structure, to slowing of blood circulation, andnecrosis of liver cells. Liver fibrosis is a dynamic process that mayprogress or regress over periods as short as months. As scar tissuebuilds up, due to inflammation and the continuance of liver injury, itcan eventually disrupt the metabolic functions of the liver. Advancedliver fibrosis can result in cirrhosis, liver failure, and portalhypertension and often requires liver transplantation (and can occurfollowing liver transplantation).

Liver fibrosis may occur in one of at least four stages. Stage 1 liverfibrosis (portal stage) is characterized by normal sized triads, portalinflammation and possible subtle bile duct damage. Granulomas may bedetected in Stage 1 liver fibrosis. Stage 2 liver fibrosis (periportalstage) may be characterized by enlarged triads, periportal fibrosisand/or inflammation. Stage 2 is characterized by the finding of aproliferation of small bile ducts. Stage 3 (septal stage) liver fibrosisis characterized by active and/or passive fibrous septa. Stage 4 ischaracterized by biliary cirrhosis and liver nodules.

Liver fibrosis may be associated with elevations of liver enzymes suchas aspartate aminotransferase (AST), alanine aminotransferase (ALT) andalkaline phosphatase (AP). Elevated liver enzymes may show up in bloodtests before any actual fibrosis occurs in response to the damage thatleads to the fibrosis. The present disclosure provides non-invasivemethods of diagnosing and prognosing liver fibrosis.

As liver functioning is impaired overt signs and symptoms may manifestincluding inflammation, pain in the area of the liver, loss of appetite,nausea and vomiting, jaundice (yellowing of the skin and the whites ofthe eyes), spider angioma, caput medusa (appearance of dilated veins onthe abdomen), and discoloration of the skin in rash-like patches. Overtsigns and/or symptoms may indicate that the scarring has progressed to apotentially dangerous level but will not occur in all cases or stages ofliver fibrosis.

Liver-fibrosis-related molecule(s): A nucleic acid or protein (orfragment thereof) whose expression is affected by liver fibrosis.Specific exemplary proteins include those listed in Tables 1, 2, 3, 5, 6and 12, as well as fragments thereof (such as the peptides in SEQ IDNOS: 1-5544), whose expression is altered (such as upregulated ordownregulated) in response to liver fibrosis (such as slow- orfast-progressing liver fibrosis). Examples of liver fibrosis-relatedmolecules whose expression is upregulated following development of liverfibrosis include those in Table 1 with a positive Log 2 fold change suchas alpha-1-antitrypsin, galectin-3-binding protein, von Willebrandfactor, complement protein C7, extracellular matrix protein 1,actin—cytoplasmic 1 (ACTB), and sulfhydryl oxidase 1, Hemoglobin subunitbeta, apoliprotein E, and sex hormone binding globulin. Specificexamples of liver fibrosis-related molecules whose expression isdownregulated following development of liver fibrosis include those inTable 1 with a negative Log 2 fold change such as prothrom bin, BCHE(Cholinesterase), Retinol binding protein 4, IGFALS, transthyretin,alpha-1-acid glycoprotein 1, alpha-1-acid glycoprotein 2, fibrinogenalpha chain, apolipoprotein C-Ill, serum albumin, Beta-Ala-Hisdipeptidase, Coagulation factor XI, Plasma kallikrein, and coagulationfactor IX (as well as nucleic acids encoding such proteins).

Liver fibrosis-related molecules also include families of proteins, suchas those associated with liver metabolism (e.g., F2, BCHE, RBP4, TTR,IGFALS, and IGFBP3), the innate immune response (e.g., CFI, C4A, C6,CBA, C7, MBL2, MASP2, and FCN3), oxidative stress (e.g., QSOX1, GPX3,and PRDX2), and liver fibrosis (e.g., F10, C5, VTN, ECM, LGALS3BP, andLUM).

Liver fibrosis-related molecules can be involved in or influenced byliver fibrosis in different ways, including causative (in that a changein a liver fibrosis-related protein leads to development of orprogression to liver fibrosis) or resultive (in that development of orprogression to liver fibrosis causes or results in a change in the liverfibrosis-related molecule).

Prognosis: To determine whether a subject will develop a disease in thefuture, such as the predisposition of a subject to develop liverfibrosis in the future, such as slow- or fast-progressing liverfibrosis.

Protein: An amino acid-based molecule as found in a mammal or othersubject. Includes full length amino acid molecules, as well as a portionof the naturally occurring full-length amino acid based molecule, suchas at least 10 contiguous amino acids of the full-length naturallyoccurring protein. A protein may accordingly be a peptide which may ormay not be found in a mammal but may instead form part of a largermolecule present naturally in a mammal. As a specific example, the“proteins listed in any of Tables 1, 2, 3, 5, 6 and 12, as well as anyof SEQ ID NOS: 1-5544” includes the full-length listed proteins,fragments of at least 10 contiguous amino acids of each of the listedproteins (such as the peptides in any of SEQ ID NOS: 1-5544), aprecursor (e.g., a pro-protein or a pre-pro-protein) of the listedproteins, or an amino-acid based metabolite of one of the listedproteins.

Sample: Biological specimens containing protein and/or nucleic acidmolecules, such as those present in peripheral blood or fractionthereof, urine, saliva, tissue biopsy (such as a liver biopsy sample),surgical specimen, fine needle aspirates, and autopsy material. In oneexample, a sample includes plasma or serum obtained from a mammaliansubject. In one example, the sample is a liquid sample.

Subject: Living multicellular vertebrate organisms having a liver. Thisterm includes both human and veterinary subjects who have a liver, suchas those that are in need of the desired diagnosis, such as diagnosis ofliver fibrosis. Examples of mammals include, but are not limited to:humans, pigs, cows, goats, cats, dogs, rabbits and mice. In one example,a subject has or is susceptible to developing liver fibrosis, such asone infected with hepatitis B or C. In one example, the subject hasreceived a liver transplant.

Therapeutically effective amount: A dose sufficient to preventadvancement, delay progression, or to cause regression of a disease, orwhich is capable of reducing symptoms caused by the disease, such asliver fibrosis. In one example, a therapeutically effective amount is anamount of a therapy sufficient to reduce inflammation in the liver,reduce liver enzyme levels (such as AST, ALT, and/or AP) and/or reducescarring of the liver by at least 10%, at least 20%, at least 50%, atleast 70%, or at least 90%. In one example, a therapeutically effectiveamount is an amount of a therapy sufficient to increase liver functionin a fibrotic liver, for example an increase of at least 10%, at least20%, at least 50%, at least 70%, or at least 90% as compared to anabsence of therapy.

Upregulated or activated: When used in reference to the expression of aprotein, refers to any process which results in an increase inproduction or expression of a protein, such as an increase of at least10%, at least 20%, at least 50%, or at least 90%, such as an at least1.2-fold, at least 1.5 fold, at least 2-fold, at least 3-fold, at least4-fold, or at least 5-fold increase. Protein upregulation includes anysignificant increase in the detectable protein in a sample. In certainexamples, the detectable protein in a sample increases by at least 10%,at least 20%, at least 50%, or at least 90%, such as 1.2-fold, at least1.5 fold, at least at least 2-fold, at least 3-fold or at least 4-fold,as compared to a control (such an amount of the detectable protein in asample from a normal subject). In one example, a control is a relativeamount of protein expression in a serum sample from a subject who doesnot have liver fibrosis. Similarly, when used in reference to theexpression of a nucleic acid, such as cDNA or mRNA, refers to anyprocess which results in an increase in production or expression of anucleic acid, such as an increase of at least 10%, at least 20%, atleast 50%, or at least 90%, such as an at least 1.2-fold, at least 1.5fold, at least 2-fold, at least 3-fold, at least 4-fold, or at least5-fold increase relative to a control.

Overview

Rapid diagnosis and prognosis of disease using less invasive, safer, andmore clinically acceptable approaches than presently employed is a goalfor medicine. While mass spectrometry (MS)-based proteomics approacheshave attempted to meet these objectives, challenges such as the enormousdynamic range of protein concentrations in clinically relevant biofluidsamples coupled with the need to address human biodiversity have slowedtheir employment. Provided herein is the use of a new instrumentalplatform that addresses these challenges by coupling technical advancesin rapid gas phase multiplexed ion mobility spectrometry (IMS)separations with liquid chromatography (LC) and MS to dramaticallyincrease measurement sensitivity and throughput. Using this LC-IMS-MSplatform, blood serum samples from 60 non-transplant and 60 post-livertransplant patients with recurrent fibrosis progression were analyzed,and relevant proteins and protein fragments identified. Themultidimensional LC-IMS-MS platform provided herein greatly improvesupon existing MS technologies in analytical sensitivity and specificity,enhances dynamic range of measurements, and provides reliableidentification and quantitation of low abundance analyte species inhighly complex biological matrices. Additionally the enhanced throughputof the new platform demonstrate the ability of this technology inclinical studies Thus, coupling ion mobility spectrometry (IMS)separation to MS can increase measurement sensitivity whilesimultaneously reducing analysis time, allowing the IMS-MS platform tobe used to identify clinically relevant proteins. These proteins andpeptide fragments can be detected in a biological sample, such as bloodor a fraction thereof, to diagnose or prognose liver fibrosis.

Based on this identification, methods, systems, and kits are providedfor diagnosing and/or prognosing liver fibrosis by analyzing theexpression of several protein markers (or their corresponding nucleicacids) in a biological sample. Methods, systems, and kits are alsoprovided for determining progression type (fast-progressing,slow-progressing and non-progressing) for subjects having liverfibrosis. Accurate detection and identification protein markers relatedto liver fibrosis, rather than more limited individual biomarkers, hasbeen demonstrated herein. Use of larger panels of protein markers canaid in the reliable prognosis, diagnosis, tracking and determination ofdisease progression. Moreover, a method for reliable identification andquantitation of low abundance liver fibrosis-related proteins has beendemonstrated. Early detection of liver fibrosis can be accomplished bycomparing the expression levels of the liver fibrosis-related proteins(or nucleic acids) to control levels reflecting average protein (ornucleic acid) levels in a normal population or in relevantsub-populations. Alternatively, an individual's protein (or nucleicacid) abundances can be compared to a baseline panel establishedspecifically for that person prior to disease onset. The method canperformed using a variety of detection methods including immunoassays ormass spectrometry (MS) approaches.

One or more steps of the disclosed methods can be performed by asuitably-programmed computer. For example, the determined level ofexpression for the at least 2 liver fibrosis-related molecules listed inany of Tables 1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS:1-5544, in a sample obtained from the subject can be inputted into acomputer or algorithm, which then generates an output, thereby analyzingthe sample. In some examples, the output (such as a visual or audibleoutput) is an indication as to whether the subject has or will likelydevelop fibrosis of the liver (such as a fast or slow-progressingfibrosis). This can allow a physician to identify those subjects whoshould receive anti-fibrosis therapy. Also provided are one or morenon-transitory computer-readable media that include computer-executableinstructions causing a computing system to perform the methods providedherein.

Systems for diagnosing or prognosing liver fibrosis in a subject arealso provided. Such systems can include a means for measuring a level ofat least two, at least 3, or at least 4 liver fibrosis-related moleculeslisted in any of Tables 1, 2, 3, 5, 6 and 12, as well as any of SEQ IDNOS: 1-5544, in a sample obtained from the subject. In some examples,such means include a mass spectrometer, ion mobility separator, liquidchromatography materials, light microscope, automated tissue or slidestainer, computer, or combinations thereof. In some examples, the systemincludes implemented rules for comparing the measured level of liverfibrosis-related molecules to one or more corresponding reference valuesor controls (for example to provide an indication as to whether there isdifferential expression). In some examples, the reference values arestored values or stored digital images. In some examples, the referencevalues are levels of liver fibrosis-related molecules measured from acontrol sample by said means for measuring. The system can also includeone or more means for implementing the rules (such as a computer oralgorithm), whereby a diagnosis or prognosis of liver fibrosis isprovided based on the measured level of liver fibrosis-related molecules(for example if differential expression is detected).

Methods for Diagnosis and Prognosis of Liver Fibrosis

The disclosure provides methods for diagnosing liver fibrosis in asubject. Diagnosis can include determining that the subject currentlyhas liver fibrosis, such as a slow-, or fast-progressing liver fibrosis.Also provided are methods for prognosing liver fibrosis in a subject.Prognosis can include determining that the subject will develop liverfibrosis in the future, such as a non-progressing, slow- orfast-progressing liver fibrosis, or include determining that currentliver fibrosis in the subject will be non-progressing, slow- orfast-progressing liver fibrosis in the future.

The disclosed methods can include detecting a plurality of liverfibrosis-related molecules (e.g., at least 2, at least 3, or at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10 proteins or corresponding nucleic acid molecules) in a sampleobtained from the subject. For example, protein expression can bedetected in a biological sample using routine methods (such asimmunoassays and spectrometry). In one example, protein expression isdetected in a sample using liquid chromatography ion mobility massspectrometry (LC-IMS-MS). The detected liver fibrosis related moleculesare compared to a control(s), wherein the control may representexpression of the same liver fibrosis-related molecules (e.g., proteins)in a subject who does not have liver fibrosis or who has non-progressingliver fibrosis. Liver fibrosis is diagnosed in the subject whendifferential expression of the two or more liver fibrosis-relatedmolecules between the sample and the control is detected or observed.Similarly, liver fibrosis is prognosed in the subject when differentialexpression of the at least two (e.g., at least 3, at least 4, at least5, at least 6, at least 7, at least 8, at least 9, or at least 10) liverfibrosis-related molecules between the sample and the control isdetected.

Various methods can be employed to select the at least two (e.g., atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, or at least 10) liver fibrosis-related molecules for inclusionin the disclosed methods, kits or other panel. In some embodiments, theat least two (e.g., at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10) liver fibrosis-relatedmolecules are selected at least in part based on having a high level ofdifferential expression such as, for example, at least 20% differentialexpression relative to a control representing the level of expression ina subject without liver fibrosis or with non-progressing liver fibrosis.In some embodiments, the at least two (e.g., at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, or at least 10)liver fibrosis-related molecules are selected at least in part based onhaving a high level of sensitivity, individually or in combination, suchas at least 90%, 95%, 96%, 97%, 98% or 99% sensitivity for the diagnosisor prognosis of liver fibrosis (such as moderate to advanced liverfibrosis). In some embodiments, the at least two (e.g., at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10) proteins are selected at least in part based on having ahigh level of specificity, individually or in combination, such as atleast 90%, 95%, 96%, 97%, 98% or 99% specificity for the diagnosis orprognosis of liver fibrosis (such as moderate to advanced liverfibrosis). In some embodiments, the at least two (e.g., at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10) liver fibrosis-related molecules are selected at least inpart based on the ability to distinguish various types and/or levels ofliver fibrosis based on the differential expression detected. Forexample, one or more of the liver fibrosis-related molecules can bechosen at least in part based on an expectation of increased expressionof the protein in fast-progressing liver fibrosis relative to a controland decreased expression of the protein relative to the control inslow-progressing liver fibrosis (e.g., where control is no liverfibrosis or non-progressing liver fibrosis).

In some examples, the method is performed at least one month, at leasttwo months, at least six months or at least one year before the onset ofclinical signs and symptoms that indicate liver fibrosis, includingsigns and symptoms indicating fast-progressing or slow-progressing liverfibrosis, regardless of cause. Specifically, the method may be performedat least one month, at least two months, at least six months or at leastone year before the onset of portal hypertension and/or clinicalmanifestations of portal hypertension such caput medusa and ascites. Insome examples, the method is performed at least one month, at least twomonths, at least six months or at least one year before the elevationabove the normal range in the subject of one, two or three of AST, ALTand alkaline phosphatase.

In various embodiments, the method detects or prognoses liver fibrosiswith a sensitivity of at least 80%, at least 85%, at least 90% or atleast 95%; and a specificity of at least 80%, at least 85%, at least 90%or at least 95%. In various embodiments, the method detects or prognosesfast-progressing liver fibrosis with a sensitivity of at least 80%, atleast 85%, at least 90% or at least 95% and a specificity of at least80%, at least 85%, at least 90% or at least 95%. In various embodiments,the method detects or prognoses slow-progressing liver fibrosis with asensitivity of at least 80%, at least 85%, at least 90% or at least 95%;and a specificity of at least 80%, at least 85%, at least 90% or atleast 95%. In some examples, differential expression of the at least two(e.g., at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10) liver fibrosis-related moleculesmay be detected as early as 1 day, as early as 2 days, as early as 5days, or as early as 10 days following the onset of pathologicalfibrotic change in the liver.

I. Exemplary Liver Fibrosis Related Molecules

This section provides descriptions for several exemplary liverfibrosis-related molecules which can be used in the disclosed methodsand kits. In some examples, the disclosed methods or systems includedetecting or measuring expression of at least 2, at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, or at least 15 ofthe liver fibrosis-related molecules listed below, such as 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14 or all 15 of the liver fibrosis-relatedmolecules listed below.

In some examples, the disclosed methods include detecting or measuringexpression of at least 2, at least 3, at least 4, or all 5 of QSOX1,ECM1, LGALS3BP, lumican and vitronectin. In some examples, the disclosedmethods include detecting or measuring expression of at least 2, atleast 3, at least 4, or all 5 of DOPA (DBH), transthyretin (TTR),cholinesterase (BCHE), retinol-binding protein (RBP4), and IGFALS. Insome examples, the disclosed methods include detecting or measuringexpression of at least 2, at least 3, at least 4 at least 5, at least 6,at least 7, at least 8, at least 9, or all 10 of QSOX1, ECM1, LGALS3BP,lumican, vitronectin, DOPA (DBH), transthyretin (TTR), BCHE, RBP4, andIGFALS. Similarly, in some examples, the disclosed kits include agentsthat permit detection or measurement of expression of at least 2, atleast 3, at least 4, or all 5 of QSOX1, ECM1, LGALS3BP, lumican andvitronectin. In some examples, the disclosed kits include agents thatpermit detection or measurement of expression of at least 2, at least 3,at least 4, or all 5 of DOPA (DBH), TTR, BCHE, RBP4, and IGFALS. In someexamples, the disclosed kits include agents that permit detection ormeasurement of expression of at least 2, at least 3, at least 4 at least5, at least 6, at least 7, at least 8, at least 9, or all 10 of QSOX1,ECM1, LGALS3BP, lumican, vitronectin, DOPA (DBH), TTR, BCHE, RBP4, andIGFALS.

A. Extracellular matrix protein 1 (ECM1) (OMIM: 602201) is anextracellular protein containing motifs with a cysteine patterncharacteristic of the cysteine pattern of the ligand-binding“double-loop” domains of the albumin protein family. This gene mapsoutside of the epidermal differentiation complex (EDC), a cluster ofthree gene families involved in epidermal differentiation. Alternativelyspliced transcript variants encoding distinct isoforms have beendescribed. Nucleic acid and protein sequences for ECM1 are publiclyavailable. For example, GENBANK® Accession Nos.: NM_(—)001202858.1 andNM_(—)001252653.1 disclose exemplary ECM1 nucleic acid sequences, andGENPEPT® Accession Nos.: NP_(—)001189787.1 (human) and NP_(—)001239582.1(mouse) disclose exemplary ECM1 protein sequences, all of which areincorporated by reference as provided by GENPEPT®/GENBANK® on Mar. 14,2013. In certain examples, ECM1 has at least 80% sequence identity, forexample at least 85%, 90%, 95%, or 98% sequence identity to a publiclyavailable ECM1 sequence, and is upregulated in the presence of liverfibrosis.

B. Prothrombin (F2) (OMIM 176930) is a protein precursor enzymaticallycleaved at two sites by activated Factor X (Xa) to produce thrombin. Inthe blood coagulation pathway, thrombin acts to convert factor XI toXIa, VIII to VIIIa, V to Va, and fibrinogen to fibrin. The F2 gene islocated on the chromosome 11 in humans (11p11-q12). Prothrombin isproduced in the liver and is post-translationally modified in a vitaminK-dependent reaction that converts ten glutamic acids on prothrombin togamma-carboxyglutamic acid (Gla). Nucleic acid and protein sequences forF2 are publicly available. For example, GENBANK®Accession Nos.:NM_(—)000506.2 (human) and NM_(—)010168.2 (mouse) are exemplary F2 acidsequences, and GENPEPT® Accession Nos.: NP_(—)000497.1 (human) andNP_(—)034298.1 (mouse) disclose exemplary F2 protein sequences, all ofwhich are incorporated by reference as provided by GENPEPT®/GENBANK® onMar. 14, 2013. In certain examples, prothrombin has at least 80%sequence identity, for example at least 85%, 90%, 95%, or 98% sequenceidentity to a publicly available prothrombin sequence, and isdownregulated in the presence of liver fibrosis.

C. Complement C4-A (C4-A) (OMIM 120810) a protein precursor forming partof the classical activation blood coagulation pathway. The protein isexpressed as a single chain precursor which is proteolytically cleavedinto a trimer of alpha, beta, and gamma chains prior to secretion. Thetrimer provides a surface for interaction between the antigen-antibodycomplex and other complement components. The alpha chain may be cleavedto release C4 anaphylatoxin, a mediator of local inflammation. This genelocalizes to the major histocompatibility complex (MHC) class III regionon chromosome 6 in humans. Nucleic acid and protein sequences for C4-Aare publicly available. For example, GENBANK® Accession Nos.:NM_(—)001252204.1 (human) and NM_(—)009780.2 (mouse) are exemplary C4-Anucleic acid sequences, and GENPEPT®/GENBANK® Accession Nos.:NP_(—)001239133.1 (human) and AAI41051.1 (mouse) disclose exemplary C4-Aprotein sequences, all of which are incorporated by reference asprovided by GENPEPT®/GENBANK® on Mar. 14, 2013. In certain examples,C4-A has at least 80% sequence identity, for example at least 85%, 90%,95%, or 98% sequence identity to a publicly available C4-A sequence, andis downregulated in the presence of liver fibrosis.

D. Sulfhydryl oxidase 1 (QSOX1) (OMIM: 603120; EC 1.8.3.2) is an enzymewhose expression is induced as fibroblasts begin to exit theproliferative cycle and enter quiescence. Nucleic acid and proteinsequences for sulfhydryl oxidase are publicly available. For example,GENBANK® Accession Nos.: NM_(—)001004128.2 (human) and NM_(—)001024945.1(mouse) are exemplary QSOX1 nucleic acid sequences, and GENPEPT®Accession Nos.: NP_(—)001004128.1 (human) and NP_(—)001020116.1 (mouse)disclose exemplary QSOX1 protein sequences, all of which areincorporated by reference as provided by GENPEPT®/GENBANK® on Mar. 14,2013. In certain examples, QSOX1 has at least 80% sequence identity, forexample at least 85%, 90%, 95%, or 98% sequence identity to a publiclyavailable QSOX1 sequence, and is upregulated in the presence of liverfibrosis.

E. Galectin-3-binding protein (LGALS3BP) (OMIM: 600626) is a proteinimplicated in modulating cell-cell and cell-matrix interactions The fulllength 90K cDNA has been localized to chromosome 17q25 in humans. Thenative protein binds specifically to a human macrophage-associatedlectin known as Mac-2 and also binds galectin 1. LGALS3BP has been foundelevated in the serum of patients with cancer and in those infected bythe human immunodeficiency virus (HIV). Nucleic acid and proteinsequences for LGALS3BP are publicly available. For example, GENBANK®Accession Nos.: NM_(—)005567.3 (human) and NM_(—)011150.2 (mouse) areexemplary LGALS3BP nucleic acid sequences, and GENPEPT® Accession Nos.:NP_(—)005558.1 (human) and NP_(—)035280.1 (mouse) disclose exemplaryLGALS3BP protein sequences, all of which are incorporated by referenceas provided by GENPEPT®/GENBANK® on Mar. 14, 2013. In certain examples,LGALS3BP has at least 80% sequence identity, for example at least 85%,90%, 95%, or 98% sequence identity to a publicly available LGALS3BPsequence, and is upregulated in liver fibrosis.

F. Coagulation Factor X (F10) (OMIM: 613872, EC 3.4.21.6) is an enzymesynthesized in the liver and requires vitamin K for its synthesis.Factor X is activated into factor Xa by both factor IX (with itscofactor, factor VIII) and factor VII with its cofactor, tissue factor.It acts by cleaving prothrombin in two places (an arg-thr and then anarg-ile bond), which yields the active thrombin. Nucleic acid andprotein sequences for Coagulation Factor X are publicly available. Forexample, GENBANK® Accession Nos.: NM_(—)000504.3 (human) andNM_(—)001242368.1 (mouse) exemplary F10 nucleic acid sequences, andGENPEPT® Accession Nos.: NP_(—)000495.1 (human) and NP_(—)001229297.1(mouse) disclose exemplary F10 protein sequences, all of which areincorporated by reference as provided by GENPEPT®/GENBANK® on Mar. 14,2013. In certain examples, F10 has at least 80% sequence identity, forexample at least 85%, 90%, 95%, or 98% sequence identity to a publiclyavailable F10 sequence, and is upregulated in the presence offast-progressing liver fibrosis but downregulated in the presence ofslow-progressing liver fibrosis.

G. Cytoskeletal β-actin (Actin—cytoplasmic 1, ACTB) (OMIM: 102630) is ahighly conserved actin protein involved in cell motility, structure andintegrity. Nucleic acid and protein sequences for ACTB are publiclyavailable. For example, GENBANK® Accession Nos.: NM_(—)001101.3 (human)and NM_(—)007393.3 (mouse) are exemplary ACTB nucleic acid sequences,and GENPEPT® Accession Nos.: NP_(—)001092.1 (human) and NP_(—)031419.1(mouse) disclose exemplary ACTB protein sequences, all of which areincorporated by reference as provided by GENPEPT®/GENBANK® on Mar. 14,2013. In certain examples, ACTB has at least 80% sequence identity, forexample at least 85%, 90%, 95%, or 98% sequence identity to a publiclyavailable ACTB sequence, and is upregulated in liver fibrosis.

H. Complement component 5 (C5) (OMIM: 120900) is a protein which playsan important role in inflammatory and cytotoxic processes. This proteinis composed of alpha and beta polypeptide chains that are linked by adisulfide bridge. An activation peptide, C5a, which is an anaphylatoxinthat possesses potent spasmogenic and chemotactic activity, is derivedfrom the alpha polypeptide via cleavage with a convertase. The C5bmacromolecular cleavage product can form a complex with the C6complement component, and this complex is the basis for formation of themembrane attack complex, which includes additional complementcomponents. Nucleic acid and protein sequences for C5 are publiclyavailable. For example, GENBANK® Accession Nos.: NM_(—)001735.2 (human)and NM_(—)010406.2 (mouse) are exemplary C5 nucleic acid sequences, andGENPEPT® Accession Nos.: NP_(—)001726.2 (human) and NP_(—)034536.1(mouse) disclose exemplary C5 protein sequences, all of which areincorporated by reference as provided by GENPEPT®/GENBANK® on Mar. 14,2013. In certain examples, C5 has at least 80% sequence identity, forexample at least 85%, 90%, 95%, or 98% sequence identity to a publiclyavailable C5 sequence, and is upregulated in the presence offast-progressing liver fibrosis but downregulated in the presence ofslow-progressing liver fibrosis.

I. Vitronectin (VTN) (OMIM: 193190) is an abundant glycoprotein found inserum and the extracellular matrix that promotes cell adhesion andspreading, inhibits the membrane-damaging effect of the terminalcytolytic complement pathway, and binds to several serpin serineprotease inhibitors. It is a secreted protein and exists in either asingle chain form or a clipped, two chain form held together by adisulfide bond. Nucleic acid and protein sequences for VTN are publiclyavailable. For example, GENBANK® Accession Nos.: NM_(—)000638.3 (human)and NM_(—)011707.2 (mouse) are exemplary VTN nucleic acid sequences, andGENPEPT® Accession Nos.: NP_(—)000629.3 (human) and NP_(—)035837.1(mouse) disclose exemplary VTN protein sequences, all of which areincorporated by reference as provided by GENPEPT®/GENBANK® on Mar. 14,2013. In certain examples, VTN has at least 80% sequence identity, forexample at least 85%, 90%, 95%, or 98% sequence identity to a publiclyavailable VTN sequence, and is up-regulated in the presence offast-progressing liver fibrosis but down-regulated in the presence ofslow-progressing liver fibrosis.

J. Lumican (LUM) (OMIM: 600616) is a protein which is the major keratansulfate proteoglycan of the cornea but is also distributed ininterstitial collagenous matrices throughout the body. Lumican mayregulate collagen fibril organization and circumferential growth,corneal transparency, and epithelial cell migration and tissue repair.Nucleic acid and protein sequences for LUM are publicly available. Forexample, GENBANK® Accession Nos.: NM_(—)002345.3 (human) andNM_(—)008524.2 (mouse) are exemplary LUM nucleic acid sequences, andGENPEPT® Accession Nos.: NP_(—)002336.1 (human) and NP_(—)032550.2(mouse) disclose exemplary LUM protein sequences, all of which areincorporated by reference as provided by GENPEPT®/GENBANK® on Mar. 14,2013. In certain examples, LUM has at least 80% sequence identity, forexample at least 85%, 90%, 95%, or 98% sequence identity to a publiclyavailable LUM sequence, and is up-regulated in the presence offast-progressing liver fibrosis but down-regulated in the presence ofslow-progressing liver fibrosis.

K. Dopamine β-hydroxylase (DBH) (OMIM: 609312) is an enzyme (oxygenase)involved in the synthesis of small-molecule neurotransmitters andconverts dopamine to norepinephrine. It is expressed in noradrenergicnerve terminals of the central and peripheral nervous systems, as wellas in chromaffin cells of the adrenal medulla. Nucleic acid and proteinsequences for DBH are publicly available. For example, GENBANK®Accession Nos.: NM_(—)000787.3 (human) and NM_(—)138942.3 (mouse) areexemplary DBH nucleic acid sequences, and GENPEPT®/GENBANK® AccessionNos.: NP_(—)000778.3 (human) and NP_(—)620392.2 (mouse) discloseexemplary DBH protein sequences, all of which are incorporated byreference as provided by GENPEPT®/GENBANK® on Mar. 14, 2013. In certainexamples, DBH has at least 80% sequence identity, for example at least85%, 90%, 95%, or 98% sequence identity to a publicly available DBHsequence, and is upregulated in the presence of liver fibrosis.

L. Transthyretin (TTR) (OMIM: 176300) is a protein and carrier ofthyroxine (T4) and retinol binding protein (when bound to retinol). Theliver secretes TTR into the blood, and the choroid plexus secretes TTRinto the cerebrospinal fluid. Nucleic acid and protein sequences for TTRare publicly available. For example, GENBANK® Accession Nos.:NM_(—)000371.3 (human) and NM_(—)013697.5 (mouse) are exemplary TTRnucleic acid sequences, and GENPEPT®/GENBANK® Accession Nos.:NP_(—)000362.1 (human) and NP_(—)038725.1 (mouse) disclose exemplary TTRprotein sequences, all of which are incorporated by reference asprovided by GENPEPT®/GENBANK® on Mar. 14, 2013. In certain examples, TTRhas at least 80% sequence identity, for example at least 85%, 90%, 95%,or 98% sequence identity to a publicly available TTR sequence, and isdownregulated in the presence of liver fibrosis.

M. Butyrylcholinesterase (BCHE) (OMIM: 177400), also known as plasmacholinesterase or pseudocholinesterase, is a non-specific cholinesteraseprotein enzyme found primarily in the liver. Nucleic acid and proteinsequences for BCHE are publicly available. For example, GENBANK®Accession Nos.: NM_(—)000055.2 (human) and NM_(—)009738.3 (mouse) areexemplary BCHE nucleic acid sequences, and GENPEPT®/GENBANK® AccessionNos.: NP_(—)000046.1 (human) and NP_(—)033868.3 (mouse) discloseexemplary BCHE protein sequences, all of which are incorporated byreference as provided by GENPEPT®/GENBANK® on Mar. 14, 2013. In certainexamples, BCHE has at least 80% sequence identity, for example at least85%, 90%, 95%, or 98% sequence identity to a publicly available BCHEsequence, and is downregulated in the presence of liver fibrosis.

N. Retinol-binding protein (RBP4) (OMIM: 180250) is a protein andspecific carrier for retinol in the blood, delivering retinol from liverstores to peripheral tissues. Nucleic acid and protein sequences forRBP4 are publicly available. For example, GENBANK® Accession Nos.:NM_(—)006744.3 (human) and NM_(—)001159487.1 (mouse) are exemplary RBP4nucleic acid sequences, and GENPEPT®/GENBANK® Accession Nos.:NP_(—)006735.2 (human) and NP_(—)001152959.1 (mouse) disclose exemplaryRBP4 protein sequences, all of which are incorporated by reference asprovided by GENPEPT®/GENBANK® on Mar. 14, 2013. In certain examples,RBP4 has at least 80% sequence identity, for example at least 85%, 90%,95%, or 98% sequence identity to a publicly available RBP4 sequence, andis downregulated in the presence of liver fibrosis.

O. Insulin-like growth factor binding protein, acid labile subunit(IGFALS) (OMIM: 601489) is a serum protein that binds insulin-likegrowth factors, increasing their half-life and vascular localization.Nucleic acid and protein sequences for IGFALS are publicly available.For example, GENBANK® Accession Nos.: NM_(—)001146006.1 (human) andNM_(—)008340.3 (mouse) are exemplary IGFALS nucleic acid sequences, andGENPEPT®/GENBANK® Accession Nos.: NP_(—)001139478.1 (human) andNP_(—)032366.2 (mouse) disclose exemplary IGFALS protein sequences, allof which are incorporated by reference as provided by GENPEPT®/GENBANK®on Mar. 14, 2013. In certain examples, IGFALS has at least 80% sequenceidentity, for example at least 85%, 90%, 95%, or 98% sequence identityto a publicly available IGFALS sequence, and is downregulated in thepresence of liver fibrosis.

II. Subjects

Liver fibrosis diagnosed or prognosed using the methods, systems, orkits herein can be due to any condition that results in cirrhosis orfibrosis, such as one or more of chronic alcohol exposure, hepatitis Bvirus (HBV) infection, non-alcoholic, steatohepatitis, hepatitis C virus(HCV) infection, Wilson's disease, alpha-1-antitrypsin deficiency,hemochromatosis, primary biliary cirrhosis, primary sclerosingcholangitis, and autoimmune hepatitis. Thus, in some examples, thesubject to be diagnosed or prognosed is one having one or more of theseconditions. In some examples, a subject having one or more of chronicalcohol exposure, hepatitis B virus infection, non-alcoholic,steatohepatitis, hepatitis C virus infection, Wilson's disease,alpha-1-antitrypsin deficiency, hemochromatosis, primary biliarycirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis isselected for analysis with the disclosed methods.

Subjects that can be diagnosed or prognosed with the disclosed methods,systems, or kits include those that have or are suspected of havingpost-transplant liver fibrosis. Thus, in some examples, the methodincludes selecting a subject who has received a liver transplant.Subjects can be any subject with a liver, such as a mammal, for examplea human or veterinary subject.

In some examples, the subject has previously received a livertransplant, and method is performed at least 10 days; at least 15 days;at least 30 days; at least 60 days; at least 90 days; at least 120 days;at least 200 days; at least 365 days; at least 2 years; or at least 3years after liver transplant.

In some examples, the subject has been infected and/or exposed to HBV orHCV. Thus, in some examples the subject to be diagnosed or prognosed isone who has received a liver transplant and has been previously infectedor exposed to HBV or HCV. For example, the disclosed methods can be isperformed at least 6 months, at least 1 year, at least 2 years, at least5 five years, at least 10 years, at least 15 years, at least 20 years,at least 25 years, or at least 30 years after the HBV or HCVexposure/infection.

In some examples, the subject is one who has been diagnosed with chronicalcoholism. Chronic alcoholism can lead to the accumulation of fattydeposits in the liver and eventual fibrosis, cirrhosis and liverfailure. In some embodiments, the subject has chronic alcoholism and themethod is performed at least 2 years, at least 3 years, at least 5years, at least 10 years, at least 15 years, at least 20 years, at least25 years, or at least 30 years after disease onset.

In some examples, the subject has alpha-antitrypsin (AAT) deficiency.Alpha-1 antitrypsin deficiency is an autosomal recessive geneticdisorder causing both lung and liver diseases. There are several formsand degrees of deficiency, principally depending on whether the suffererhas one or two copies of the affected gene. The most common genotype ofAAT deficiency is genotype PiZZ, which encodes mutant AAT, referred toas Z protein (ATZ). The fundamental pathological process of the AATdeficiency is the accumulation of mutant AAT as polymers withinhepatocytes. The resultant low levels of AAT in the serum, result inlung damage by proteinases, and eventually emphysema. The proteinaccumulation in hepatocytes may play a role in the development of liverdiseases, including chronic hepatitis, liver fibrosis, cirrhosis andhepatocellular carcinoma.

In some examples, the subject has hemochromatosis. Hereditaryhemochromatosis (HH), is characterized by increased intestinalabsorption of iron, which leads to deposition of iron in the body, e.g.,in multiple organs such as liver, pancreas, heart and other organs.Excess iron deposition, if left untreated, causes tissue damage andfibrosis with irreversible damage to various organs, e.g. ironoverloaded organs, e.g. endocrine dysfunction or hepatic fibrosis andcirrhosis.

In some examples, the subject has primary biliary cirrhosis (PBC). PBCis an autoimmune disease of the liver marked by the progressivedestruction of small bile ducts of the liver, with the intralobularducts affected early in the disease. When the ducts are damaged, bilemay build up in the liver and over time damage the tissue and lead toscarring, fibrosis and cirrhosis. Those affected by PBC may present withsymptoms of fatigue, pruritis (itchiness), jaundice and xanthomas.

In some examples, the subject has primary sclerosing cholangitis (PSC).PSC is a disease of the bile ducts that causes inflammation andsubsequent obstruction of the bile ducts at an intra- and extra-hepaticlevel. The inflammation impedes the flow of bile to the gut which canlead to liver fibrosis, liver cirrhosis, liver failure and liver cancer.More than 80% of those with PSC have ulcerative colitis. Symptoms of PSCmay be similar to those of PBC and can include pruritis, jaundice,fatigue, and malabsorption. Approximately 80% subjects with PSC haveperinuclear anti-neutrophil cytoplasmic antibodies (p-ANCA).

In some examples, the subject has Wilson's disease. Wilson's disease,also known as hepatolenticular degeneration, is a rare inheritedsystemic disorder of copper metabolism. Individuals with Wilson'sdisease are unable to excrete copper into their bile, thus, copperbegins to accumulate in the liver. Wilson's disease may cause acute orchronic hepatitis (inflammation of the liver) or cirrhosis (severe liverdisease) due to a progressive loss of liver function. The degree ofliver involvement is variable and may range from mild elevations ofcertain liver enzymes to complete liver failure. Symptoms associatedwith Wilson's disease include fatigue, anorexia, weight loss,generalized weakness, ascites and abdominal swelling, or jaundice. Othersymptoms include enlargement of the liver (hepatomegaly), spleen(splenomegaly), or both (hepatosplenomegaly).

In some examples, the subject has nonalcoholic fatty liver disease(NAFLD) such as nonalcoholic steatohepatitis (NASH). Subjects with NAFLDhave no drinking habit (less than 20 g daily), however give histologicalfindings characterized by hepatic fatty deposition reminiscent of thosefound in alcoholic hepatic damage. The disease is regarded as aphenotype in the liver of metabolic syndrome accompanying obesity. NAFLDis divided into simple fatty liver and NASH. NASH is a progressivedisease that frequently accompanies fibrosis and has been known toprogress to hepatic cirrhosis and hepatic cancer. NAFLD may be suspectedin subjects with high levels of triglyceride in the blood and may bediagnosed by abdominal ultrasonography and CT.

In some examples, the subject has autoimmune hepatitis. Autoimmunehepatitis is a disease of the liver that occurs when the body's immunesystem attacks cells of the liver. Anomalous presentation of humanleukocyte antigen (HLA) class II on the surface of hepatocytes possiblydue to a genetic predisposition or acute liver infection, causes acell-mediated immune response against the body's own liver. Thisabnormal immune response results in inflammation of the liver, which canlead to further complications, including fibrosis and cirrhosis.

III. Differential Expression

Detecting differential expression can include measuring a change in geneor protein expression, such as a change in amount (e.g., qualitative orquantitative) of one or more liver fibrosis-related molecules (e.g.,proteins, peptides, or nucleic acids). For example, measurement ordetection of expression can be absolute or relative, and in someexamples is quantified. In various embodiments, differential expressionincludes a detectable increase or decrease of at least 1.1-fold; atleast 1.15-fold; at least 1.2-fold; at least 1.25-fold; at least1.3-fold; at least 1.4-fold; at least 1.5-fold; at least 1.6-fold; atleast 1.7-fold; at least 2-fold; at least 2.5-fold; at least 3-fold; atleast 3.5-fold; or at least 4-fold relative to a control. In someembodiments, differential expression includes a detectable increase ordecrease of at least 10%; at least 20%; at least 30%; at least 40%; atleast 50%; at least 60%; at least 70%; at least 80%; at least 90%; atleast 100%; at least 125%; at least 150%; at least 200%; at least 300%,or at least 400% relative to a control. In certain embodiments,differential expression of at least two (e.g., at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10, such as 2, 5, 10 or 15) liver fibrosis-related molecules between thesample and the control is detected, wherein differential expression ofat least 1.2 fold is detected for at least 50%; at least 75%; at least80%; at least 90%; or all of the at least two (e.g., at least 3, atleast 4, at least 5, at least 6, at least 7, at least 8, at least 9, orat least 10, such as 2, 5, 10 or 15) liver fibrosis-related moleculestested.

In one embodiment, the differential expression detected for each of theat least two (e.g., at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10, such as 2, 5, 10 or 15)liver fibrosis-related molecules is compared to the expecteddifferential expression for each liver fibrosis-related molecule for asubject with liver fibrosis to determine whether the detecteddifferential expressions indicate a diagnosis of liver fibrosis. In someembodiments, the level of differential expression required to prognosethe development of liver fibrosis will vary from the level required todiagnose the presence of liver fibrosis. In one embodiment, the requireddifferential expression of the at least two (e.g., at least 3, at least4, at least 5, at least 6, at least 7, at least 8, at least 9, or atleast 10, such as 2, 5, 10 or 15) liver fibrosis-related molecules forprognosis of liver fibrosis is smaller than that required for diagnosis.In certain embodiments, the time at which differential expression of theat least two (e.g., at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10, such as 2, 5, 10 or 15)liver fibrosis-related molecules is measured is earlier for prognosisthan for diagnosis. In various embodiments, differential expression ismeasured at least 10 days, at least 30 days, at least 90 days, at least2 months, at least 3 months, at least 6 months, at least 1 year, atleast 2 years, at least 3 years, at least 5 years or at least 10 yearsearlier for prognosis of liver fibrosis than for diagnosis.

In certain embodiments, the at least two (e.g., at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, or at least10, such as 2, 5, 10 or 15) liver fibrosis-related molecules areselected for their suitability for prognosis. In some embodiments, theat least two (e.g., at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10, such as 2, 5, 10 or 15)liver fibrosis-related molecules are selected for prognosis on the basisof their showing differential expression at an early time point ascompared to non-selected proteins. Exemplary markers that can be usedfor prognosis include VTN, LUM, and F10. In some embodiments, the atleast two (e.g., at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or at least 10, such as 2, 5, 10 or 15)liver fibrosis-related molecules are selected for diagnosis also on thebasis of their showing differential expression at an earlier time pointas compared to non-selected proteins. In various embodiments, asignificant differential expression in the amount of the at least two(e.g., at least 3, at least 4, at least 5, at least 6, at least 7, atleast 8, at least 9, or at least 10, such as 2, 5, 10 or 15) liverfibrosis related molecules in the serum or plasma (or other sample) of asubject compared to the amount of the same liver fibrosis relatedmolecule found in a control is at least at a 1.1-fold, at least 1.2fold, at least 1.5 fold, at least 2-fold, at least 3-fold, at least4-fold or greater increase or decrease. Substantial differentialexpression, including overexpression or underexpression, of the at leasttwo (e.g., at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, or at least 10, such as 2, 5, 10 or 15) or moreliver fibrosis related molecule(s) can be indicative of the presence ofliver fibrosis such as fast-progressing liver fibrosis orslow-progressing liver fibrosis.

In some examples, the control is a reference value or range of valuesrepresenting a quantitative or qualitative amount of expression (e.g.,protein, peptide, or nucleic acid) expected in a subject without liverfibrosis. In certain embodiments, the control is a predicted referencevalue or range of values representing a quantitative or qualitativeamount of expression (e.g., protein, peptide, or nucleic acid) expectedfor the subject based on his/her personal characteristics such as healthstatus, genetics and demographic information. In certain embodiments,the detected expression (e.g., protein, peptide, or nucleic acid) for asubject is compared to the protein expression for a matched controlsubject such as a control subject without liver fibrosis or withnon-progressing liver fibrosis. In some embodiments, the control is areference value or range of values representing a quantitative orqualitative amount of protein expression expected in a subject withnon-progressing liver fibrosis. In some embodiments, the control is areference value or range of values representing a quantitative orqualitative amount of protein expression expected in a subject withslow-progressing liver fibrosis. In some embodiments, the control is areference value or range of values representing a quantitative orqualitative amount of protein expression expected in a subject withfast-progressing liver fibrosis. In some embodiments, the control is areference value or range of values representing a quantitative orqualitative amount of protein expression expected in a subject withoutliver fibrosis but with HCV or HBV infection.

Tables 1-3 and 5-6 below provide differential expression information forspecific liver fibrosis-related proteins. In one example, at least 10 orat least 15 of the peptides shown in any of SEQ ID NOS: 1-5544 aredetected as an indication of protein expression. One skilled in the artwill appreciate that as an alternative to detecting protein expression,expression of the corresponding nucleic acid molecule can be detected.In some examples, expression of the plurality of proteins or nucleicacids is detected simultaneously or contemporaneously in a sample.Furthermore, due to multiplexing technologies, one will appreciate thatmultiple samples, for example from different subjects, can be analyzedsimultaneously or contemporaneously.

Table 1 provides directionality and relative amounts of differentialexpression detected for liver fibrosis-related proteins in subjectshaving post-transplant liver fibrosis (fast-progressing orslow-progressing) relative to matched controls representing the amountof protein expression in a subject with non-progressing liver fibrosis.Differential expression for the proteins ranged from 1.1-fold to morethan 3-fold. Thus, expression of two or more of the liverfibrosis-related proteins (or corresponding peptides or nucleic acidmolecules) in Table 1 can be used to diagnose or prognose liver fibrosisin a subject, such as by detecting expression of at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 31, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, at least 63, at least 64, at least65, at least 66, at least 67, at least 68, at least 69, at least 70, atleast 71, at least 72, at least 73, at least 74, at least 75, at least76, at least 77, at least 78, at least 79, at least 80, at least 81, atleast 82, at least 83, at least 84, at least 85, at least 86, at least87, at least 88, at least 89, at least 90, at least 91, at least 92, atleast 93, at least 94, at least 95, at least 96, at least 97, at least98, at least 99, at least 100, at least 101, at least 102, at least 103,at least 104, at least 105, at least 106, at least 107, at least 108, atleast 109, at least 110, at least 111, at least 112, at least 113, atleast 114, at least 115, at least 116, at least 117, at least 118, atleast 119, at least 120, at least 121, at least 122, at least 123, atleast 124, at least 125, at least 126, at least 127, at least 128, atleast 129, at least 130, at least 131, at least 132, at least 133, atleast 134, at least 135, or all 136 of the proteins (or correspondingpeptides or nucleic acid molecules) in Table 1. For example, if at leasttwo of the liver fibrosis-related molecules in Table 1 (such as at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 31, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 41, at least42, at least 43, at least 44, at least 45, at least 46, at least 47, atleast 48, at least 49, at least 50, at least 51, at least 52, at least53, at least 54, at least 55, at least 56, at least 57, at least 58, atleast 59, at least 60, at least 61, at least 62, at least 63, at least64, at least 65, at least 66, at least 67, at least 68, at least 69, atleast 70, at least 71, at least 72, at least 73, at least 74, at least75, at least 76, at least 77, at least 78, at least 79, at least 80, atleast 81, at least 82, at least 83, at least 84, at least 85, at least86, at least 87, at least 88, at least 89, at least 90, at least 91, atleast 92, at least 93, at least 94, at least 95, at least 96, at least97, at least 98, at least 99, at least 100, at least 101, at least 102,at least 103, at least 104, at least 105, at least 106, at least 107, atleast 108, at least 109, at least 110, at least 111, at least 112, atleast 113, at least 114, at least 115, at least 116, at least 117, atleast 118, at least 119, at least 120, at least 121, at least 122, atleast 123, at least 124, at least 125, at least 126, at least 127, atleast 128, at least 129, at least 130, at least 131, at least 132, atleast 133, at least 134, at least 135, or all 136 of the proteins) showthe relative amount and direction of differential expression in the testsample, relative to a control (such as an amount of protein expressionexpected in a subject or population of subjects having non-progressingliver fibrosis), this results in a diagnosis of liver fibrosis, or aprognosis that the subject will develop liver fibrosis in the future.

Also shown in Table 1 is that 63 of the proteins also exhibiteddifferential expression in non-transplant patients with liver fibrosis(see Table 5). Additionally, after excluding 4 proteins which decreasein SP, but increase in FP, >91% are observed with common abundancedirectionality with the non-transplant results (Table 12), providingstrong orthogonal validation of the proteins based upon both transplantand non-transplant patients. 26 proteins (Ig kappa chain V-III regionVG, Ig kappa chain V-III region HAH, Ig lambda chain V-III region LOI,IGHA1, IGHG1, IGHG2, IGKC, IGKV1-5, IGLC1, FCGR3A, C7, HBB, LGALS3BP,MBL2, SERPINA1, SHBG, CNDP1, CNDP1, ALB, APOC3, C4A, FGA, ORM1, ORM2,TTR, and FN1) showed at least a 1.5-fold change in both slow- andfast-progressing liver fibrosis, and 10 proteins (Ig kappa chain V-IIIregion VG, Ig kappa chain V-III region HAH, Ig lambda chain V-III regionLOI, IGHA1, IGHG1, IGHG2, IGKV1-5, SERPINA1, APOC3, and TTR) showed morethan 2-fold differential expression in both slow- and fast-progressingliver fibrosis. Thus in some examples, these 26 proteins or 10 proteinsare used in the methods and systems provided herein to diagnose orprognose liver fibrosis.

TABLE 1 Changes in 136 Liver Fibrosis Related Proteins in Slow- and/orFast-Progressing Liver Fibrosis relative to matched controlsrepresenting protein expression in a subject with non-progressing liverfibrosis Protein and UniProt Accession Up- Down- Fold-Change vs. GeneNumber gulated regulated Control Proteins significantly upregulated inthe Slow and Fast Progressors (SP & FP) compared to their matchedcontrols PTPRG P23470 Receptor-type tyrosine- X At least 1.2-foldprotein phosphatase gamma QSOX1 O00391 Sulfhydryl oxidase 1 X At least1.4-fold GPX3 P22352 Glutathione peroxidase 3 X At least 1.2-fold .P04433 Ig kappa chain V-III region VG X At least 2-fold . P18135 Igkappa chain V-III region X At least 2.2-fold HAH . P80748 Ig lambdachain V-III region X At least 2.1-fold LOI IGHA1 P01876 Ig alpha-1 chainC region X At least 2.8-fold IGHG1 P01857 Ig gamma-1 chain C region X Atleast 2.2-fold IGHG2 P01859 Ig gamma-2 chain C region X At least2.4-fold IGHM P01871 Ig mu chain C region X At least 1.6-fold IGKCP01834 Ig kappa chain C region X At least 1.8-fold IGKV1-5 P01602 Igkappa chain V-I region X At least 2-fold HK102 IGLC1 P01842 Ig lambdachain C regions X At least 1.8-fold FCGR3A P08637 Low affinityimmunoglobulin X At least 1.5-fold gamma Fc region receptor III-A A2MP01023 Alpha-2-macroglobulin X At least 1.4-fold ACTB Q96HG5 Actin,cytoplasmic 1 X At least 1.2-fold AFM P43652 Afamin X At least 1.3-foldAHSG P02765 Alpha-2-HS-glycoprotein X At least 1.3-fold ALCAM Q13740CD166 antigen X At least 1.3-fold APOB Q13787 Apolipoprotein B-100 X Atleast 1.2-fold APOE P02649 Apolipoprotein E X At least 1.3-fold C7P10643 Complement component C7 X At least 1.8-fold CLU P10909 ClusterinX At least 1.2-fold ECM1 Q8IZ60 Extracellular matrix protein 1 X Atleast 1.3-fold HBB Q549N7 Hemoglobin subunit beta X At least 1.5-foldITIH3 Q06033 Inter-alpha-trypsin inhibitor X At least 1.2-fold heavychain H3 LGALS3BP Q08380 Galectin-3-binding protein X At least 1.5-foldMBL2 P11226 Mannose-binding protein C X At least 1.6-fold SELL P14151L-selectin X At least 1.2-fold SERPINA1 Q86U18 Alpha-1-antitrypsin X Atleast 3-fold SERPINA6 P08185 Corticosteroid-binding X At least 1.2-foldglobulin SHBG P04278 Sex hormone-binding globulin X At least 1.5-foldVCAM1 P19320 Vascular cell adhesion protein 1 X At least 1.3-fold VWFP04275 von Willebrand factor X At least 1.3-fold Proteins significantlydownregulated in the Slow and Fast Progressors (SP & FP) compared totheir matched controls F2 P00734 Prothrombin X At least 1.2-fold CFBQ9BX92 Complement factor B X At least 1.1-fold CFI P05156 Complementfactor I X At least 1.1-fold C1S Q9UCV4 Complement C1s X At least1.2-fold subcomponent C1R P00736 Complement C1r X At least 1.2-foldsubcomponent KLKB1 P03952 Plasma kallikrein X At least 1.3-fold F11P03951 Coagulation factor XI X At least 1.3-fold F9 P00740 Coagulationfactor IX X At least 1.2-fold CNDP1 Q96KN2 Beta-Ala-His dipeptidase X Atleast 1.8-fold BCHE P06276 Cholinesterase X At least 1.3-fold ALB Q9P157Serum albumin X At least 1.9-fold APCS P02743 Serum amyloid P-componentX At least 1.3-fold APOA4 P06727 Apolipoprotein A-IV X At least 1.2-foldAPOC3 P02656 Apolipoprotein C-III X At least 2.2-fold APOH P02749Beta-2-glycoprotein 1 X At least 1.3-fold AZGP1 P25311Zinc-alpha-2-glycoprotein X At least 1.2-fold C4A Q5JQM8 Complement C4-AX At least 1.5-fold C6 P13671 Complement component C6 X At least1.2-fold C8A P07357 Complement component C8 X At least 1.3-fold alphachain C8B P07358 Complement component C8 X At least 1.3-fold beta chainC8G P07360 Complement component C8 X At least 1.2-fold gamma chain C9P02748 Complement component C9 X At least 1.2-fold FGA P02671 Fibrinogenalpha chain X At least 1.8-fold HPX P02790 Hemopexin X At least 1.2-foldHRG P04196 Histidine-rich glycoprotein X At least 1.3-fold IGFALS P35858Insulin-like growth factor- X At least 1.3-fold binding protein complexacid labile subunit ITIH1 P19827 Inter-alpha-trypsin inhibitor X Atleast 1.3-fold heavy chain H1 ITIH2 P19823 Inter-alpha-trypsin inhibitorX At least 1.1-fold heavy chain H2 ITIH4 Q14624 Inter-alpha-trypsininhibitor X At least 1.1-fold heavy chain H4 ORM1 P02763 Alpha-1-acidglycoprotein 1 X At least 1.8-fold ORM2 P19652 Alpha-1-acid glycoprotein2 X At least 1.5-fold PRG4 Q92954 Proteoglycan 4 X At least 1.2-foldRBP4 P02753 Retinol-binding protein 4 X At least 1.1-fold SERPINA3Q6NSC9 Alpha-1-antichymotrypsin X At least 1.1-fold SERPINC1 Q9UC78Antithrombin-III X At least 1.1-fold SERPIND1 P05546 Heparin cofactor 2X At least 1.2-fold TTR P02766 Transthyretin X At least 2.2-foldProteins showing opposing significant changes in the Slow and FastProgressors (SP & FP) compared to matched controls F10 P00742Coagulation factor X X X Slow- Progressing: At least 1.2-fold (decrease)Fast- Progressing: At least 1.2 fold (increase) FN1 Q9HAP3 Fibronectin XX Slow- Progressing: At least 1.5-fold (decrease) Fast- Progressing: Atleast 1.5-fold (increase) C5 P01031 Complement C5 X X Slow- Progressing:At least 1.1-fold (increase) Fast- Progressing: At least 1.2-fold(increase) SERPINA4 P29622 Kallistatin X X Slow- Progressing: At least1.2-fold (decrease) Fast- Progressing: At least 1.3-fold (increase) VTNP04004 Vitronectin X X Slow- Progressing: At least 1.1-fold (decrease)Fast- Progressing: At least 1.2-fold (increase) LUM P51884 Lumican X XSlow- Progressing: At least 2.6-fold (decrease) Fast- Progressing: Atleast 1.1-fold (increase) Proteins significantly increasing/decreasingonly in Slow Progressors (SP) compared to their matched controls HBA1P69905 Hemoglobin subunit alpha X At least 2.1-fold SERPING1 Q96FE0Plasma protease C1 inhibitor X At least 1.4-fold . P01594 Ig kappa chainV-I region AU X At least 3-fold . P01614 Ig kappa chain V-II region CumX At least 3.2-fold IGKV4-1 P06312 Ig kappa chain V-IV region X At least3.3-fold PGLYRP2 Q96PD5 N-acetylmuramoyl-L-alanine X At least 1.2 foldamidase BTD P43251 Biotinidase X At least 1.2 fold PLG P00747Plasminogen X At least 1.1 fold PROC P04070 Vitamin K-dependent proteinC X At least 1.6 fold C1RL Q9NZP8 Complement C1r X At least 1.2 foldsubcomponent-like protein CPN1 P15169 Carboxypeptidase N catalytic X Atleast 1.1 fold chain CPB2 Q96IY4 Carboxypeptidase B2 X At least 1.2 foldAPOA1 P02647 Apolipoprotein A-I* X At least 1.5 fold C4B Q9UIP5Complement C4-B X At least 1.5 fold GC P02774 Vitamin D-binding proteinX At least 1.1 fold GP5 P40197 Platelet glycoprotein V X At least 1.2fold HP P00738 Haptoglobin X At least 1.4 fold IGFBP3 P17936Insulin-like growth factor- X At least 1.3 fold binding protein 3* KNG1P01042 Kininogen-1 X At least 1.3 fold PROS1 P07225 Vitamin K-dependentprotein S X At least 1.3 fold SERPINA10 Q9UK55 Protein Z-dependentprotease X At least 1.4 fold inhibitor SERPINF1 P36955 Pigmentepithelium-derived X At least 1.2 fold factor SERPINF2 P08697Alpha-2-antiplasmin X At least 1.1 fold THBS1 P07996 Thrombospondin-1 XAt least 1.4 fold Proteins significantly changing only in FastProgressors (FP) compared to their matched controls PTGDS P41222Prostaglandin-H2 D-isomerase X At least 1.2-fold CA1 P00915 Carbonicanhydrase 1 X At least 2.1-fold ALDOB P05062 Fructose-bisphosphate X Atleast 1.7-fold aldolase B C2 P06681 Complement C2 X At least 1.2-foldF12 P00748 Coagulation factor XII X At least 1.2-fold MASP2 Q9H498Mannan-binding lectin serine X At least 1.2-fold protease 2 HABP2 Q14520Hyaluronan-binding protein 2 X At least 1.4-fold ANPEP P15144Aminopeptidase N X At least 1.4-fold ENPP2 Q9UCR2 Ectonucleotide X Atleast 1.5-fold pyrophosphatase/phosphodiesterase family member 2 CPP00450 Ceruloplasmin X At least 1.1-fold PRDX2 P32119 Peroxiredoxin-2 XAt least 1.6-fold A1BG P04217 Alpha-1B-glycoprotein* X At least 1.1-foldTGFBI Q53XM1 Transforming growth factor- X At least 1.4-foldbeta-induced protein ig-h3 CD14 P08571 Monocyte differentiation X Atleast 1.3-fold antigen CD14 C3 P01024 Complement C3 X At least 1.3-foldENG P17813 Endoglin X At least 1.3-fold PROCR Q9UNN8 Endothelial proteinC X At least 1.2-fold receptor FCGBP Q9Y6R7 IgGFc-binding protein X Atleast 1.4-fold FCN3 O75636 Ficolin-3 X At least 1.3-fold FETUB Q9UGM5Fetuin-B X At least 1.3-fold MST1 P26927 Hepatocyte growth factor-like XAt least 1.4-fold protein MCAM P43121 Cell surface glycoprotein X Atleast 1.2-fold MUC18 NRP1 O14786 Neuropilin-1 X At least 1.3-fold SPP2Q13103 Secreted phosphoprotein 24 X At least 1.4-fold SERPINA7 P05543Thyroxine-binding globulin X At least 1.5-fold VASN Q6EMK4 Vasorin X Atleast 1.2-fold COL18A1 Q96T70 Collagen alpha-1(XVIII) chain X At least1.8-fold PVR P15151 Poliovirus receptor X At least 1.6-fold APOA2 P02652Apolipoprotein A-II X At least 1.8-fold PI16 Q6UXB8 Peptidase inhibitor16 X At least 1.9-fold H6PD O95479 GDH/6PGL endoplasmic X At least1.5-fold bifunctional protein AGT P01019 Angiotensinogen X At least1.3-fold CFH Q9NU86 Complement factor H X At least 1.2-fold CRP P02741C-reactive protein X At least 1.6-fold GSN P06396 Gelsolin X At least1.3-fold

Table 2 provides directionality and relative amounts of differentialexpression detected for liver fibrosis-related proteins in subjectshaving post-transplant liver fibrosis which showed differentialexpression in fast-progressing liver fibrosis relative to matchedcontrols representing the amount of protein expression in a subject withnon-progressing liver fibrosis. Differential expression for the proteinsranged from 1.1-fold to more than 3-fold. The fold-change observed forthose proteins showing differential expression in fast-progressing liverfibrosis is also provided in Table 2. A large group of the proteins (40)showed more than a 1.5-fold change in fast-progressing liver fibrosis,and 13 proteins showed more than a 2-fold change. Thus in some examples,these 40 proteins or 13 proteins are used in the methods and systemsprovided herein to diagnose or prognose fast-progressing liver fibrosis.41 of the proteins either do not show differential expression inslow-progressing liver fibrosis or show differential expression in theopposite direction. Of these 41 proteins, 12 had more than a 1.5-foldchange in fast-progressing liver fibrosis. Thus in some examples, these12 proteins are used in the methods and systems provided herein todiagnose or prognose fast-progressing liver fibrosis.

Thus, expression of two or more of the liver fibrosis-related proteins(or corresponding peptides or nucleic acid molecules) in Table 2 can beused to diagnose or prognose fast-progressing liver fibrosis in asubject, such as by detecting expression of at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 31, at least 31, at least 32, atleast 33, at least 34, at least 35, at least 36, at least 37, at least38, at least 39, at least 40, at least 41, at least 42, at least 43, atleast 44, at least 45, at least 46, at least 47, at least 48, at least49, at least 50, at least 51, at least 52, at least 53, at least 54, atleast 55, at least 56, at least 57, at least 58, at least 59, at least60, at least 61, at least 62, at least 63, at least 64, at least 65, atleast 66, at least 67, at least 68, at least 69, at least 70, at least71, at least 72, at least 73, at least 74, at least 75, at least 76, atleast 77, at least 78, at least 79, at least 80, at least 81, at least82, at least 83, at least 84, at least 85, at least 86, at least 87, atleast 88, at least 89, at least 90, at least 91, at least 92, at least93, at least 94, at least 95, at least 96, at least 97, at least 98, atleast 99, at least 100, at least 101, at least 102, at least 103, atleast 104, at least 105, at least 106, at least 107, at least 108, atleast 109, at least 110, at least 111, or all 112 of the proteins (orcorresponding peptides or nucleic acid molecules) in Table 2. Forexample, if at least two of the liver fibrosis-related molecules inTable 2 (such as at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, at least 31, at least 31, at least 32, at least 33, at least34, at least 35, at least 36, at least 37, at least 38, at least 39, atleast 40, at least 41, at least 42, at least 43, at least 44, at least45, at least 46, at least 47, at least 48, at least 49, at least 50, atleast 51, at least 52, at least 53, at least 54, at least 55, at least56, at least 57, at least 58, at least 59, at least 60, at least 61, atleast 62, at least 63, at least 64, at least 65, at least 66, at least67, at least 68, at least 69, at least 70, at least 71, at least 72, atleast 73, at least 74, at least 75, at least 76, at least 77, at least78, at least 79, at least 80, at least 81, at least 82, at least 83, atleast 84, at least 85, at least 86, at least 87, at least 88, at least89, at least 90, at least 91, at least 92, at least 93, at least 94, atleast 95, at least 96, at least 97, at least 98, at least 99, at least100, at least 101, at least 102, at least 103, at least 104, at least105, at least 106, at least 107, at least 108, at least 109, at least110, at least 111, or all 112 of the proteins) show the relative amountand direction of differential expression in the test sample, relative toa control (such as an amount of protein expression expected in a subjector population of subjects having non-progressing liver fibrosis), thisresults in a diagnosis of fast-progressing liver fibrosis, or aprognosis that the subject will develop a fast-progressing liverfibrosis in the future.

Similarly, expression of two or more of the liver fibrosis-relatedpeptides in any of SEQ ID NOS: 1281-2633 can be used to diagnose orprognose fast-progressing liver fibrosis in a subject. Thus, expressionof 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612,613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640,641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682,683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696,697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710,711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724,725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738,739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752,753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780,781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794,795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808,809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836,837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850,851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864,865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878,879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892,893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906,907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920,921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934,935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948,949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962,963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976,977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990,991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003,1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015,1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027,1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039,1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051,1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063,1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075,1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087,1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099,1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111,1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123,1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135,1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147,1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159,1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171,1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183,1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195,1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207,1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219,1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231,1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243,1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255,1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267,1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279,1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291,1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303,1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315,1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327,1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339,1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351,1352, or 1353 of the liver fibrosis-related peptides in any of SEQ IDNOS: 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291,1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303,1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315,1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327,1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339,1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351,1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363,1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375,1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387,1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399,1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411,1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423,1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435,1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447,1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459,1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471,1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483,1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495,1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507,1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519,1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531,1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543,1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555,1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567,1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579,1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591,1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603,1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615,1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627,1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639,1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651,1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663,1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675,1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687,1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699,1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711,1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723,1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735,1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747,1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759,1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771,1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783,1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795,1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807,1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819,1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831,1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843,1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855,1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867,1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879,1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891,1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901, 1902, 1903,1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915,1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927,1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939,1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951,1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963,1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975,1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985, 1986, 1987,1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997, 1998, 1999,2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009, 2010, 2011,2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021, 2022, 2023,2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033, 2034, 2035,2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045, 2046, 2047,2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057, 2058, 2059,2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069, 2070, 2071,2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081, 2082, 2083,2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093, 2094, 2095,2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105, 2106, 2107,2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117, 2118, 2119,2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129, 2130, 2131,2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141, 2142, 2143,2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153, 2154, 2155,2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165, 2166, 2167,2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176, 2177, 2178, 2179,2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189, 2190, 2191,2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201, 2202, 2203,2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214, 2215,2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226, 2227,2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238, 2239,2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250, 2251,2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261, 2262, 2263,2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274, 2275,2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286, 2287,2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298, 2299,2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310, 2311,2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322, 2323,2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334, 2335,2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346, 2347,2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358, 2359,2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370, 2371,2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382, 2383,2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394, 2395,2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406, 2407,2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418, 2419,2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430, 2431,2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442, 2443,2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454, 2455,2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466, 2467,2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478, 2479,2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490, 2491,2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502, 2503,2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514, 2515,2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 2526, 2527,2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537, 2538, 2539,2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549, 2550, 2551,2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561, 2562, 2563,2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573, 2574, 2575,2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585, 2586, 2587,2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597, 2598, 2599,2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609, 2610, 2611,2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621, 2622, 2623,2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, and 2633, can beused to diagnose or prognose fast-progressing liver fibrosis in asubject. For example, if two or more of SEQ ID NOS: 1281-1869 aredownregualted relative to a control (such as an amount of proteinexpression expected in a subject or population of subjects havingnon-progressing liver fibrosis), such as two or more of any of SEQ IDNOS: 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291,1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303,1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315,1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327,1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339,1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351,1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363,1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375,1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387,1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397, 1398, 1399,1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409, 1410, 1411,1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421, 1422, 1423,1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433, 1434, 1435,1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445, 1446, 1447,1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457, 1458, 1459,1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469, 1470, 1471,1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481, 1482, 1483,1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493, 1494, 1495,1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505, 1506, 1507,1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517, 1518, 1519,1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529, 1530, 1531,1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541, 1542, 1543,1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553, 1554, 1555,1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565, 1566, 1567,1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577, 1578, 1579,1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589, 1590, 1591,1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601, 1602, 1603,1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613, 1614, 1615,1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625, 1626, 1627,1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637, 1638, 1639,1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649, 1650, 1651,1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661, 1662, 1663,1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673, 1674, 1675,1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685, 1686, 1687,1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697, 1698, 1699,1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709, 1710, 1711,1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721, 1722, 1723,1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733, 1734, 1735,1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745, 1746, 1747,1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757, 1758, 1759,1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769, 1770, 1771,1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781, 1782, 1783,1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793, 1794, 1795,1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805, 1806, 1807,1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817, 1818, 1819,1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829, 1830, 1831,1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843,1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855,1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867,1868, and 1869, this can be used to diagnose or prognosefast-progressing liver fibrosis in a subject. In some examples,downregulation is a decrease of at least 0.5 fold, at least 0.8 fold, atleast 1-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, atleast 3.5-fold, at least 4-fold or at least 4.5-fold. In addition, iftwo or more of SEQ ID NOS: 1870-2633 are upregualted relative to acontrol (such as an amount of protein expression expected in a subjector population of subjects having non-progressing liver fibrosis), suchas two or more of any of SEQ ID NOS: 1870, 1871, 1872, 1873, 1874, 1875,1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887,1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899,1900, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911,1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923,1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935,1936, 1937, 1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947,1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959,1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971,1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983,1984, 1985, 1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995,1996, 1997, 1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007,2008, 2009, 2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019,2020, 2021, 2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031,2032, 2033, 2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043,2044, 2045, 2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055,2056, 2057, 2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067,2068, 2069, 2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079,2080, 2081, 2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091,2092, 2093, 2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103,2104, 2105, 2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115,2116, 2117, 2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127,2128, 2129, 2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139,2140, 2141, 2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151,2152, 2153, 2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163,2164, 2165, 2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175,2176, 2177, 2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187,2188, 2189, 2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199,2200, 2201, 2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211,2212, 2213, 2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223,2224, 2225, 2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235,2236, 2237, 2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247,2248, 2249, 2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259,2260, 2261, 2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271,2272, 2273, 2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283,2284, 2285, 2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295,2296, 2297, 2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307,2308, 2309, 2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319,2320, 2321, 2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331,2332, 2333, 2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343,2344, 2345, 2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355,2356, 2357, 2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367,2368, 2369, 2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379,2380, 2381, 2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391,2392, 2393, 2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403,2404, 2405, 2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415,2416, 2417, 2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427,2428, 2429, 2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439,2440, 2441, 2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451,2452, 2453, 2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463,2464, 2465, 2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475,2476, 2477, 2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487,2488, 2489, 2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499,2500, 2501, 2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511,2512, 2513, 2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523,2524, 2525, 2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535,2536, 2537, 2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547,2548, 2549, 2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559,2560, 2561, 2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571,2572, 2573, 2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583,2584, 2585, 2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595,2596, 2597, 2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607,2608, 2609, 2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619,2620, 2621, 2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631,2632, and 2633, this can be used to diagnose or prognosefast-progressing liver fibrosis in a subject. In some examples, theupregulation is an increase of at least 0.5 fold, at least 1-fold, atleast 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, orat least 3.3-fold.

In a specific example, expression of two or more of the liverfibrosis-related proteins (or corresponding peptides or nucleic acidmolecules) labeled under “Proteins showing significant differentialexpression only in Fast-Progressing Liver Fibrosis” in Table 2 can beused to diagnose or prognose fast-progressing liver fibrosis in asubject, such as by detecting expression of at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, at least 24, at least 25, at least 26, at least27, at least 28, at least 29, at least 31, at least 31, at least 32, atleast 33, at least 34, or all 35 such proteins (or correspondingpeptides or nucleic acid molecules) in Table 2. For example, if at leasttwo of the liver fibrosis-related molecules in Table 2 (such as at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 31, at least31, at least 32, at least 33, at least 34, or all 35 labeled under“Proteins showing significant differential expression only inFast-Progressing Liver Fibrosis”) show the relative amount and directionof differential expression in the test sample, relative to a control(such as an amount of protein expression expected in a subject orpopulation of subjects having non-progressing liver fibrosis), thisresults in a diagnosis of fast-progressing liver fibrosis, or aprognosis that the subject will develop a fast-progressing liverfibrosis in the future.

In another example, expression of two or more (e.g., at least 3, atleast 4) of the liver fibrosis-related proteins (or correspondingpeptides or nucleic acid molecules) labeled under “Proteins showingsignificant differential expression in opposite directions for Fast andSlow-Progressing Liver Fibrosis” in Table 2 can be used to diagnose orprognose fast-progressing liver fibrosis in a subject, such as bydetecting expression of at least 3, at least 4, at least 5 or all 6 suchproteins (or corresponding peptides or nucleic acid molecules) labeledunder “Proteins showing significant differential expression in oppositedirections for Fast and Slow-Progressing Liver Fibrosis” in Table 2. Forexample, if at least two (e.g., at least 3, at least 4,) of the liverfibrosis-related molecules in Table 2, such as at least 5 or all 6 ofthe proteins labeled under “Proteins showing significant differentialexpression in opposite directions for Fast and Slow-Progressing LiverFibrosis” show the relative amount and direction of differentialexpression (i.e., up-regulation) in the test sample, relative to acontrol (such as an amount of protein expression expected in a subjector population of subjects having non-progressing liver fibrosis), thisresults in a diagnosis of fast-progressing liver fibrosis, or aprognosis that the subject will develop a fast-progressing liverfibrosis in the future.

TABLE 2 112 Liver Fibrosis Related Proteins with Differential Expressionin Post-Transplant Fast-Progressing Liver Fibrosis relative to matchedcontrols representing protein expression in a subject withnon-progressing liver fibrosis Protein and UniProt Accession Up- Down-Fold-Change Gene Number regulated regulated vs. Control PTPRG P23470Receptor-type tyrosine- X At least 1.2- protein phosphatase gamma foldQSOX1 O00391 Sulfhydryl oxidase 1 X At least 1.7- fold GPX3 P22352Glutathione peroxidase 3 X At least 1.3- fold . P04433 Ig kappa chainV-III region VG X At least 3.1- fold . P18135 Ig kappa chain V-IIIregion X At least 2.3- HAH fold . P80748 Ig lambda chain V-III region XAt least 2.1- LOI fold IGHA1 P01876 Ig alpha-1 chain C region X At least2.8- fold IGHG1 P01857 Ig gamma-1 chain C region X At least 2.2- foldIGHG2 P01859 Ig gamma-2 chain C region X At least 2.4- fold IGHM P01871Ig mu chain C region X At least 1.6- fold IGKC P01834 Ig kappa chain Cregion X At least 1.8- fold IGKV1-5 P01602 Ig kappa chain V-I region XAt least 2-fold HK102 IGLC1 P01842 Ig lambda chain C regions X At least1.8- fold FCGR3A P08637 Low affinity immunoglobulin X At least 1.8-gamma Fc region receptor III-A fold A2M P01023 Alpha-2-macroglobulin XAt least 1.5- fold ACTB Q96HG5 Actin, cytoplasmic 1 X At least 1.2- foldAFM P43652 Afamin X At least 1.3- fold AHSG P02765Alpha-2-HS-glycoprotein X At least 1.5- fold ALCAM Q13740 CD166 antigenX At least 1.5- fold APOB Q13787 Apolipoprotein B-100 X At least 1.2-fold APOE P02649 Apolipoprotein E X At least 1.3- fold C7 P10643Complement component C7 X At least 2.1- fold CLU P10909 Clusterin X Atleast 1.2- fold ECM1 Q8IZ60 Extracellular matrix protein 1 X At least1.3- fold HBB Q549N7 Hemoglobin subunit beta X At least 1.5- fold ITIH3Q06033 Inter-alpha-trypsin inhibitor X At least 1.2- heavy chain H3 foldLGALS3BP Q08380 Galectin-3-binding protein X At least 2.2- fold MBL2P11226 Mannose-binding protein C X At least 1.6- fold SELL P14151L-selectin X At least 1.3- fold SERPINA1 Q86U18 Alpha-1-antitrypsin X Atleast 3-fold SERPINA6 P08185 Corticosteroid-binding X At least 1.2-globulin fold SHBG P04278 Sex hormone-binding globulin X At least 1.6-fold VCAM1 P19320 Vascular cell adhesion protein 1 X At least 1.4- foldVWF P04275 von Willebrand factor X At least 1.3- fold F2 P00734Prothrombin X At least 1.3- fold CFB Q9BX92 Complement factor B X Atleast 1.2- fold CFI P05156 Complement factor I X At least 1.1- fold C1SQ9UCV4 Complement C1s X At least 1.2- subcomponent fold C1R P00736Complement C1r X At least 1.2- subcomponent fold KLKB1 P03952 Plasmakallikrein X At least 1.3- fold F11 P03951 Coagulation factor XI X Atleast 1.3- fold F9 P00740 Coagulation factor IX X At least 1.2- foldCNDP1 Q96KN2 Beta-Ala-His dipeptidase X At least 1.8- fold BCHE P06276Cholinesterase X At least 1.3- fold ALB Q9P157 Serum albumin X At least2.8- fold APCS P02743 Serum amyloid P-component X At least 1.3- foldAPOA4 P06727 Apolipoprotein A-IV X At least 1.2- fold APOC3 P02656Apolipoprotein C-III X At least 2.4- fold APOH P02749Beta-2-glycoprotein 1 X At least 1.3- fold AZGP1 P25311Zinc-alpha-2-glycoprotein X At least 1.2- fold C4A Q5JQM8 ComplementC4-A X At least 1.5- fold C6 P13671 Complement component C6 X At least1.3- fold C8A P07357 Complement component C8 X At least 1.4- alpha chainfold C8B P07358 Complement component C8 X At least 1.3- beta chain foldC8G P07360 Complement component C8 X At least 1.2- gamma chain fold C9P02748 Complement component C9 X At least 1.2- fold FGA P02671Fibrinogen alpha chain X At least 1.8- fold HPX P02790 Hemopexin X Atleast 1.2- fold HRG P04196 Histidine-rich glycoprotein X At least 1.3-fold IGFALS P35858 Insulin-like growth factor- X At least 1.3- bindingprotein complex acid labile fold subunit ITIH1 P19827Inter-alpha-trypsin inhibitor X At least 1.3- heavy chain H1 fold ITIH2P19823 Inter-alpha-trypsin inhibitor X At least 1.1- heavy chain H2 foldITIH4 Q14624 Inter-alpha-trypsin inhibitor X At least 1.1- heavy chainH4 fold ORM1 P02763 Alpha-1-acid glycoprotein 1 X At least 1.8- foldORM2 P19652 Alpha-1-acid glycoprotein 2 X At least 1.5- fold PRG4 Q92954Proteoglycan 4 X At least 1.2- fold RBP4 P02753 Retinol-binding protein4 X At least 1.1- fold SERPINA3 Q6NSC9 Alpha-1-antichymotrypsin X Atleast 1.2- fold SERPINC1 Q9UC78 Antithrombin-III X At least 1.2- foldSERPIND1 P05546 Heparin cofactor 2 X At least 1.2- fold TTR P02766Transthyretin X At least 1.3- fold Proteins showing significantdifferential expression in opposite directions for Fast andSlow-Progressing Liver Fibrosis F10 P00742 Coagulation factor X X Atleast 1.2 fold FN1 Q9HAP3 Fibronectin X At least 1.5- fold C5 P01031Complement C5 X At least 1.2- fold SERPINA4 P29622 Kallistatin X Atleast 1.3- fold VTN P04004 Vitronectin X At least 1.2- fold LUM P51884Lumican X At least 1.1- fold Proteins showing significant differentialexpression only in Fast-Progressing Liver Fibrosis PTGDS P41222Prostaglandin-H2 D-isomerase X At least 1.2- fold CA1 P00915 Carbonicanhydrase 1 X At least 2.1- fold ALDOB P05062 Fructose-bisphosphate X Atleast 1.7- aldolase B fold C2 P06681 Complement C2 X At least 1.2- foldF12 P00748 Coagulation factor XII X At least 1.2- fold MASP2 Q9H498Mannan-binding lectin serine X At least 1.2- protease 2 fold HABP2Q14520 Hyaluronan-binding protein 2 X At least 1.4- fold ANPEP P15144Aminopeptidase N X At least 1.4- fold ENPP2 Q9UCR2 Ectonucleotide X Atleast 1.5- pyrophosphatase/phosphodiesterase fold family member 2 CPP00450 Ceruloplasmin X At least 1.1- fold PRDX2 P32119 Peroxiredoxin-2 XAt least 1.6- fold A1BG P04217 Alpha-1B-glycoprotein* X At least 1.1-fold TGFBI Q53XM1 Transforming growth factor- X At least 1.4-beta-induced protein ig-h3 fold CD14 P08571 Monocyte differentiation XAt least 1.3- antigen CD14 fold C3 P01024 Complement C3 X At least 1.3-fold ENG P17813 Endoglin X At least 1.3- fold PROCR Q9UNN8 Endothelialprotein C X At least 1.2- receptor fold FCGBP Q9Y6R7 IgGFc-bindingprotein X At least 1.4- fold FCN3 O75636 Ficolin-3 X At least 1.3- foldFETUB Q9UGM5 Fetuin-B X At least 1.3- fold MST1 P26927 Hepatocyte growthfactor-like X At least 1.4- protein fold MCAM P43121 Cell surfaceglycoprotein X At least 1.2- MUC18 fold NRP1 O14786 Neuropilin-1 X Atleast 1.3- fold SPP2 Q13103 Secreted phosphoprotein 24 X At least 1.4-fold SERPINA7 P05543 Thyroxine-binding globulin X At least 1.5- foldVASN Q6EMK4 Vasorin X At least 1.2- fold COL18A1 Q96T70 Collagenalpha-1(XVIII) chain X At least 1.8- fold PVR P15151 Poliovirus receptorX At least 1.6- fold APOA2 P02652 Apolipoprotein A-II X At least 1.8-fold PI16 Q6UXB8 Peptidase inhibitor 16 X At least 1.9- fold H6PD O95479GDH/6PGL endoplasmic X At least 1.5- bifunctional protein fold AGTP01019 Angiotensinogen X At least 1.3- fold CFH Q9NU86 Complement factorH X At least 1.2- fold CRP P02741 C-reactive protein X At least 1.6-fold GSN P06396 Gelsolin X At least 1.3- fold

Table 3 provides directionality and relative amounts of differentialexpression detected for liver fibrosis-related proteins in subjectshaving post-transplant liver fibrosis which showed differentialexpression in slow-progressing liver fibrosis relative to matchedcontrols representing the amount of protein expression in a subject withnon-progressing liver fibrosis. Differential expression for the proteinsranged from 1.1-fold to more than 3-fold. Thus, expression of two ormore of the liver fibrosis-related proteins (or corresponding peptidesor nucleic acid molecules) in Table 3 can be used to diagnose orprognose slow-progressing liver fibrosis in a subject, such as bydetecting expression of at least 3, at least 4, at least 5, at least 6,at least 7, at least 8, at least 9, at least 10, at least 11, at least12, at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, at least 31, at least 31, at least 32, at least 33, at least34, at least 35, at least 36, at least 37, at least 38, at least 39, atleast 40, at least 41, at least 42, at least 43, at least 44, at least45, at least 46, at least 47, at least 48, at least 49, at least 50, atleast 51, at least 52, at least 53, at least 54, at least 55, at least56, at least 57, at least 58, at least 59, at least 60, at least 61, atleast 62, at least 63, at least 64, at least 65, at least 66, at least67, at least 68, at least 69, at least 70, at least 71, at least 72, atleast 73, at least 74, at least 75, at least 76, at least 77, at least78, at least 79, at least 80, at least 81, at least 82, at least 83, atleast 84, at least 85, at least 86, at least 87, at least 88, at least89, at least 90, at least 91, at least 92, at least 93, at least 94, atleast 95, at least 96, at least 97, at least 98, at least 99, at least100, or all 101 of the proteins (or corresponding peptides or nucleicacid molecules) in Table 3. For example, if at least two of the liverfibrosis-related molecules in Table 3 (such as at least 3, at least 4,at least 5, at least 6, at least 7, at least 8, at least 9, at least 10,at least 11, at least 12, at least 13, at least 14, at least 15, atleast 16, at least 17, at least 18, at least 19, at least 20, at least21, at least 22, at least 23, at least 24, at least 25, at least 26, atleast 27, at least 28, at least 29, at least 31, at least 31, at least32, at least 33, at least 34, at least 35, at least 36, at least 37, atleast 38, at least 39, at least 40, at least 41, at least 42, at least43, at least 44, at least 45, at least 46, at least 47, at least 48, atleast 49, at least 50, at least 51, at least 52, at least 53, at least54, at least 55, at least 56, at least 57, at least 58, at least 59, atleast 60, at least 61, at least 62, at least 63, at least 64, at least65, at least 66, at least 67, at least 68, at least 69, at least 70, atleast 71, at least 72, at least 73, at least 74, at least 75, at least76, at least 77, at least 78, at least 79, at least 80, at least 81, atleast 82, at least 83, at least 84, at least 85, at least 86, at least87, at least 88, at least 89, at least 90, at least 91, at least 92, atleast 93, at least 94, at least 95, at least 96, at least 97, at least98, at least 99, at least 100, or all 101 of the proteins) show therelative amount and direction of differential expression in the testsample, relative to a control (such as an amount of protein expressionexpected in a subject or population of subjects having non-progressingliver fibrosis), this results in a diagnosis of slow-progressing liverfibrosis, or a prognosis that the subject will develop aslow-progressing liver fibrosis in the future.

In a specific example, expression of two or more of the liverfibrosis-related proteins (or corresponding peptides or nucleic acidmolecules) labeled under “Proteins showing significant differentialexpression only in Slow-Progressing Liver Fibrosis” in Table 3 can beused to diagnose or prognose slow-progressing liver fibrosis in asubject, such as by detecting expression of at least 3, at least 4, atleast 5, at least 6, at least 7, at least 8, at least 9, at least 10, atleast 11, at least 12, at least 13, at least 14, at least 15, at least16, at least 17, at least 18, at least 19, at least 20, at least 21, atleast 22, at least 23, or all 24 such proteins (or correspondingpeptides or nucleic acid molecules) in Table 3. For example, if at leasttwo of the liver fibrosis-related molecules in Table 3 (such as at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, or all 24, such as 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or24, labeled under “Proteins showing significant differential expressiononly in Slow-Progressing Liver Fibrosis”) show the relative amount anddirection of differential expression in the test sample, relative to acontrol (such as an amount of protein expression expected in a subjector population of subjects having non-progressing liver fibrosis), thisresults in a diagnosis of slow-progressing liver fibrosis, or aprognosis that the subject will develop a slow-progressing liverfibrosis in the future.

In another example, expression of two or more of the liverfibrosis-related proteins (or corresponding peptides or nucleic acidmolecules) labeled under “Proteins showing significant differentialexpression in opposite directions for Fast and Slow-Progressing LiverFibrosis” in Table 3 can be used to diagnose or prognoseslow-progressing liver fibrosis in a subject, such as by detectingexpression of at least 3, at least 4, at least 5 or all 6 such proteins,such as 3, 4, 5, or 6 of the proteins (or corresponding peptides ornucleic acid molecules) labeled under “Proteins showing significantdifferential expression in opposite directions for Fast andSlow-Progressing Liver Fibrosis” in Table 3. For example, if at leasttwo of the liver fibrosis-related molecules in Table 3, such as at least3, at least 4, at least 5 or all 6 of the proteins labeled under“Proteins showing significant differential expression in oppositedirections for Fast and Slow-Progressing Liver Fibrosis” show therelative amount and direction of differential expression (i.e.,down-regulation) in the test sample, relative to a control (such as anamount of protein expression expected in a subject or population ofsubjects having non-progressing liver fibrosis), this results in adiagnosis of slow-progressing liver fibrosis, or a prognosis that thesubject will develop a slow-progressing liver fibrosis in the future.

The fold-change relative to controls observed for the proteinsdemonstrating differential expression in slow-progressing liver fibrosisis provided in Table 3. A large group of the proteins (38) showed morethan a 1.5-fold change in slow-progressing liver fibrosis, and 21 ofthese proteins showed more than a 2-fold change. Thus in some examples,these 38 proteins or 21 proteins are used in the methods and systemsprovided herein to diagnose or prognose slow-progressing liver fibrosis.30 of the proteins listed with differential expression inslow-progressing liver fibrosis either did not show differentialexpression in fast-progressing liver fibrosis or exhibited differentialexpression in the opposite direction. Of these 30 proteins, 7 had morethan a 1.5-fold change in slow-progressing liver fibrosis. Thus in someexamples, these 7 proteins are used in the methods and systems providedherein to diagnose or prognose slow-progressing liver fibrosis.

Similarly, expression of two or more of the liver fibrosis-relatedpeptides in any of SEQ ID NOS: 1-1280 can be used to diagnose orprognose slow-progressing liver fibrosis in a subject. Thus, expressionof 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612,613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640,641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682,683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696,697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710,711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724,725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738,739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752,753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780,781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794,795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808,809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836,837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850,851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864,865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878,879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892,893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906,907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920,921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934,935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948,949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962,963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976,977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990,991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003,1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015,1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027,1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039,1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051,1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063,1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075,1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087,1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099,1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111,1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123,1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135,1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147,1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159,1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171,1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183,1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195,1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207,1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219,1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231,1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243,1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255,1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267,1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279,or 1280 of the liver fibrosis-related peptides in any of SEQ ID NOS: 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108,109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122,123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136,137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150,151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164,165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178,179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192,193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206,207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234,235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248,249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276,277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290,291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304,305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318,319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332,333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346,347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360,361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374,375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388,389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402,403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416,417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430,431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444,445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458,459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472,473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500,501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514,515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528,529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542,543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556,557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570,571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584,585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598,599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612,613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626,627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640,641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654,655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668,669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682,683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696,697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709, 710,711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723, 724,725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737, 738,739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751, 752,753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765, 766,767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780,781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794,795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808,809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822,823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836,837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850,851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864,865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878,879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892,893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906,907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920,921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934,935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948,949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962,963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976,977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990,991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003,1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015,1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027,1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039,1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051,1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063,1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075,1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087,1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099,1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111,1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123,1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135,1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147,1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159,1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171,1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183,1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195,1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207,1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219,1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231,1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243,1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255,1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267,1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279,and 1280, can be used to diagnose or prognose slow-progressing liverfibrosis in a subject. For example, if two or more of SEQ ID NOS:768-1280 are upregualted relative to a control (such as an amount ofprotein expression expected in a subject or population of subjectshaving non-progressing liver fibrosis), such as two or more of any ofSEQ ID NOS: 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779,780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793,794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807,808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821,822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849,850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863,864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877,878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891,892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905,906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919,920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933,934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947,948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961,962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989,990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002,1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014,1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026,1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038,1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050,1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062,1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074,1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086,1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098,1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110,1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122,1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134,1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146,1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158,1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170,1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182,1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194,1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206,1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218,1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230,1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242,1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254,1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266,1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278,1279, and 1280, this can be used to diagnose or prognoseslow-progressing liver fibrosis in a subject. In some examples, theupregulation is an increase of at least 0.1 fold, at least 0.5-fold, atleast 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, atleast 3-fold, or at least 3.1-fold. In addition, if two or more of SEQID NOS: 1-767 are downregualted relative to a control (such as an amountof protein expression expected in a subject or population of subjectshaving non-progressing liver fibrosis), such as two or more of any ofSEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343,344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399,400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455,456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469,470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483,484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497,498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511,512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525,526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553,554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567,568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581,582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609,610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651,652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665,666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693,694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707,708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721,722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735,736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749,750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763,764, 765, 766, and 767, this can be used to diagnose or prognoseslow-progressing liver fibrosis in a subject. In some examples, thedownregulation is a decrease of at least 0.5 fold, at least 0.8 fold, atleast 1-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, atleast 3.5-fold, or at least 4-fold.

TABLE 3 101 Liver Fibrosis Related Proteins with Differential Expressionin Post-Transplant Slow-Progressing Liver Fibrosis relative to matchedcontrols representing protein expression in a subject withnon-progressing liver fibrosis Up- Down- Fold-Change vs. Gene Proteinand UniProt Accession Number regulated regulated Control PTPRG P23470Receptor-type tyrosine-protein X At least 1.3-fold phosphatase gammaQSOX1 O00391 Sulfhydryl oxidase 1 X At least 1.4-fold GPX3 P22352Glutathione peroxidase 3 X At least 1.2-fold . P04433 Ig kappa chainV-III region VG X At least 2 fold . P18135 Ig kappa chain V-III regionHAH X At least 3.2-fold . P80748 Ig lambda chain V-III region LOI X Atleast 2.6-fold IGHA1 P01876 Ig alpha-1 chain C region X At least4.9-fold IGHG1 P01857 Ig gamma-1 chain C region X At least 3.6-foldIGHG2 P01859 Ig gamma-2 chain C region X At least 2.8-fold IGHM P01871Ig mu chain C region X At least 3.0-fold IGKC P01834 Ig kappa chain Cregion X At least 4.3-fold IGKV1-5 P01602 Ig kappa chain V-I regionHK102 X At least 2.3-fold IGLC1 P01842 Ig lambda chain C regions X Atleast 4.2-fold FCGR3A P08637 Low affinity immunoglobulin X At least1.5-fold gamma Fc region receptor III-A A2M P01023 Alpha-2-macroglobulinX At least 1.4-fold ACTB Q96HG5 Actin, cytoplasmic 1 X At least 1.4-foldAFM P43652 Afamin X At least 1.4-fold AHSG P02765Alpha-2-HS-glycoprotein X At least 1.3-fold ALCAM Q13740 CD166 antigen XAt least 1.3-fold APOB Q13787 Apolipoprotein B-100 X At least 1.4-foldAPOE P02649 Apolipoprotein E X At least 1.5-fold C7 P10643 Complementcomponent C7 X At least 1.8-fold CLU P10909 Clusterin X At least1.3-fold ECM1 Q8IZ60 Extracellular matrix protein 1 X At least 1.3-foldHBB Q549N7 Hemoglobin subunit beta X At least 2.3-fold ITIH3 Q06033Inter-alpha-trypsin inhibitor X At least 1.2-fold heavy chain H3LGALS3BP Q08380 Galectin-3-binding protein X At least 1.5-fold MBL2P11226 Mannose-binding protein C X At least 1.7-fold SELL P14151L-selectin X At least 1.2-fold SERPINA1 Q86U18 Alpha-1-antitrypsin X Atleast 3-fold SERPINA6 P08185 Corticosteroid-binding globulin X At least1.2-fold SHBG P04278 Sex hormone-binding globulin X At least 1.5-foldVCAM1 P19320 Vascular cell adhesion protein 1 X At least 1.3-fold VWFP04275 von Willebrand factor X At least 1.3-fold F2 P00734 Prothrombin XAt least 1.2-fold CFB Q9BX92 Complement factor B X At least 1.1-fold CFIP05156 Complement factor I X At least 1.2-fold C1S Q9UCV4 Complement C1sX At least 1.3-fold subcomponent C1R P00736 Complement C1r X At least1.2-fold subcomponent KLKB1 P03952 Plasma kallikrein X At least 1.3-foldF11 P03951 Coagulation factor XI X At least 1.5-fold F9 P00740Coagulation factor IX X At least 1.4-fold CNDP1 Q96KN2 Beta-Ala-Hisdipeptidase X At least 1.8-fold BCHE P06276 Cholinesterase X At least1.5-fold ALB Q9P157 Serum albumin X At least 1.9-fold APCS P02743 Serumamyloid P-component X At least 1.5-fold APOA4 P06727 Apolipoprotein A-IVX At least 1.1-fold APOC3 P02656 Apolipoprotein C-III X At least2.2-fold APOH P02749 Beta-2-glycoprotein 1 X At least 1.4-fold AZGP1P25311 Zinc-alpha-2-glycoprotein X At least 1.2-fold C4A Q5JQM8Complement C4-A X At least 1.6-fold C6 P13671 Complement component C6 XAt least 1.2-fold C8A P07357 Complement component C8 X At least 1.3-foldalpha chain C8B P07358 Complement component C8 X At least 1.4-fold betachain C8G P07360 Complement component C8 X At least 1.2-fold gamma chainC9 P02748 Complement component C9 X At least 1.2-fold FGA P02671Fibrinogen alpha chain X At least 4.0-fold HPX P02790 Hemopexin X Atleast 1.3-fold HRG P04196 Histidine-rich glycoprotein X At least1.3-fold IGFALS P35858 Insulin-like growth factor- X At least 1.5-foldbinding protein complex acid labile subunit ITIH1 P19827Inter-alpha-trypsin inhibitor X At least 1.4-fold heavy chain H1 ITIH2P19823 Inter-alpha-trypsin inhibitor X At least 1.2-fold heavy chain H2ITIH4 Q14624 Inter-alpha-trypsin inhibitor X At least 1.2-fold heavychain H4 ORM1 P02763 Alpha-1-acid glycoprotein 1 X At least 2.3-foldORM2 P19652 Alpha-1-acid glycoprotein 2 X At least 2.1-fold PRG4 Q92954Proteoglycan 4 X At least 1.4-fold RBP4 P02753 Retinol-binding protein 4X At least 1.1-fold SERPINA3 Q6NSC9 Alpha-1-antichymotrypsin X At least1.1-fold SERPINC1 Q9UC78 Antithrombin-III X At least 1.1-fold SERPIND1P05546 Heparin cofactor 2 X At least 1.4-fold TTR P02766 Transthyretin XAt least 2.2-fold Proteins showing significant differential expressionin opposite directions for Fast and Slow-Progressing Liver Fibrosis F10P00742 Coagulation factor X X At least 1.2-fold FN1 Q9HAP3 Fibronectin XAt least 1.5-fold C5 P01031 Complement C5 X At least 1.1-fold SERPINA4P29622 Kallistatin X At least 1.2-fold VTN P04004 Vitronectin X At least1.1-fold LUM P51884 Lumican X At least 2.6-fold Proteins showingsignificant differential expression only in Slow-Progressing LiverFibrosis HBA1 P69905 Hemoglobin subunit alpha X At least 2.1-foldSERPING1 Q96FE0 Plasma protease C1 inhibitor X At least 1.4-fold .P01594 Ig kappa chain V-I region AU X At least 3-fold . P01614 Ig kappachain V-II region Cum X At least 3.2-fold IGKV4-1 P06312 Ig kappa chainV-IV region X At least 3.3-fold PGLYRP2 Q96PD5N-acetylmuramoyl-L-alanine X At least 1.2 fold amidase BTD P43251Biotinidase X At least 1.2 fold PLG P00747 Plasminogen X At least 1.1fold PROC P04070 Vitamin K-dependent protein C X At least 1.6 fold C1RLQ9NZP8 Complement C1r X At least 1.2 fold subcomponent-like protein CPN1P15169 Carboxypeptidase N catalytic X At least 1.1 fold chain CPB2Q96IY4 Carboxypeptidase B2 X At least 1.2 fold APOA1 P02647Apolipoprotein A-I* X At least 1.5 fold C4B Q9UIP5 Complement C4-B X Atleast 1.5 fold GC P02774 Vitamin D-binding protein X At least 1.1 foldGP5 P40197 Platelet glycoprotein V X At least 1.2 fold HP P00738Haptoglobin X At least 1.4 fold IGFBP3 P17936 Insulin-like growthfactor- X At least 1.3 fold binding protein 3* KNG1 P01042 Kininogen-1 XAt least 1.3 fold PROS1 P07225 Vitamin K-dependent protein S X At least1.3 fold SERPINA10 Q9UK55 Protein Z-dependent protease X At least 1.4fold inhibitor SERPINF1 P36955 Pigment epithelium-derived X At least 1.2fold factor SERPINF2 P08697 Alpha-2-antiplasmin X At least 1.1 foldTHBS1 P07996 Thrombospondin-1 X At least 1.4 fold

Table 4 summarizes the results in Tables 1-3. As indicated in Table 4,77 proteins showed differential expression in both fast- andslow-progressing liver fibrosis (post-transplant subjects) with thedifferential expression having the same directionality for both groupsin 71 out of 77 of the proteins. 34 proteins increased in the slowprogressors (SP) and fast progressors (FP) compared to their matchedcontrols, 37 proteins decreased in the SP and FP compared to theirmatched controls, 5 proteins only increased in the SP compared to theirmatched controls, 30 proteins only increased in the FP, 19 proteins onlydecreased in the SP, 5 proteins only decreased in the FP and 6 proteinsincreased in the FP and decreased in the SP.

TABLE 4 Summary of Transplant Patients with Fibrosis FP SP SlowProgressors (SP) 34↑ 37↓ 5↑ 19↓ Fast Progressors (FP) 30↑ 5↓ 6 (↑FP↓SP)Total Significantly Changing Proteins 136

Table 5 provides directionality of differential expression for liverfibrosis-related proteins in non-transplant subjects having advancedliver fibrosis relative to controls with Stage 0-1 fibrosis. 40% of theproteins (31 out of 78 proteins) were up-regulated in the non-transplantsubjects having advanced liver fibrosis and 60% of the proteins (47 outof 78 proteins) were down-regulated. 81% of the proteins in Table 5 (63out of 78 proteins) also showed differential expression in thetransplant group (i.e., appear in Table 1) with the majority (43 out of78 proteins) being proteins showing differential expression in bothslow- and fast-progressing post-transplant subjects (i.e., proteinsappearing in both Table 2 and Table 3).

As discussed above, after excluding 4 proteins which decrease in SP, butincrease in FP, >91% of the proteins with differential expression in thetransplant group and non-transplant group are observed with commonabundance directionality, providing a strong orthogonal validation ofthe proteins based upon both transplant and non-transplant studies. Moreextensive expression data, including data for specific peptides, isfound in the Examples section below. Thus, expression of two or more ofthe liver fibrosis-related proteins (or corresponding peptides ornucleic acid molecules) in Table 5 can be used to diagnose or prognoseliver fibrosis in a subject, such as by detecting expression of at least3, at least 4, at least 5, at least 6, at least 7, at least 8, at least9, at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 16, at least 17, at least 18, at least 19, at least20, at least 21, at least 22, at least 23, at least 24, at least 25, atleast 26, at least 27, at least 28, at least 29, at least 31, at least31, at least 32, at least 33, at least 34, at least 35, at least 36, atleast 37, at least 38, at least 39, at least 40, at least 41, at least42, at least 43, at least 44, at least 45, at least 46, at least 47, atleast 48, at least 49, at least 50, at least 51, at least 52, at least53, at least 54, at least 55, at least 56, at least 57, at least 58, atleast 59, at least 60, at least 61, at least 62, at least 63, at least64, at least 65, at least 66, at least 67, at least 68, at least 69, atleast 70, at least 71, at least 72, at least 73, at least 74, at least75, at least 76, at least 77, or all 78 of the proteins (orcorresponding peptides or nucleic acid molecules) in Table 5. Forexample, if at least two of the liver fibrosis-related molecules inTable 2 (such as at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, at least 31, at least 31, at least 32, at least 33, at least34, at least 35, at least 36, at least 37, at least 38, at least 39, atleast 40, at least 41, at least 42, at least 43, at least 44, at least45, at least 46, at least 47, at least 48, at least 49, at least 50, atleast 51, at least 52, at least 53, at least 54, at least 55, at least56, at least 57, at least 58, at least 59, at least 60, at least 61, atleast 62, at least 63, at least 64, at least 65, at least 66, at least67, at least 68, at least 69, at least 70, at least 71, at least 72, atleast 73, at least 74, at least 75, at least 76, at least 77, or all 78of the proteins) show the relative amount and direction of differentialexpression in the test sample, relative to a control (such as an amountof protein expression expected in a subject or population of subjectshaving non-progressing liver fibrosis), this results in a diagnosis ofliver fibrosis, or a prognosis that the subject will develop liverfibrosis in the future.

TABLE 5 78 Liver Fibrosis Related Proteins having DifferentialExpression in Non-Transplant Stage 4-6 Liver Fibrosis subjects relativeto Controls representing protein expression in a Non-Transplant Stage0-1 Liver Fibrosis subject. Direction of Differential Gene ProteinExpression QSOX1 O00391 Sulfhydryl oxidase 1 ↑ IGLC1 P01842 Ig lambdachain C regions ↑ FCGR3A P08637 Low affinity immunoglobulin ↑ gamma Fcregion receptor III-A A2M P01023 Alpha-2-macroglobulin ↑ AFM P43652Afamin ↑ ALCAM Q13740 CD166 antigen ↑ APOB* Q13787 Apolipoprotein B-100↓ C7 P10643 Complement component C7 ↑ CLU P10909 Clusterin ↓ ECM1 Q8IZ60Extracellular matrix protein 1 ↑ ITIH3 Q06033 Inter-alpha-trypsininhibitor heavy ↑ chain H3 LGALS3BP Q08380 Galectin-3-binding protein ↑VCAM1 P19320 Vascular cell adhesion protein 1 ↑ VWF P04275 vonWillebrand factor ↑ F2 P00734 Prothrombin ↓ CFI P05156 Complement factorI ↓ KLKB1 P03952 Plasma kallikrein ↓ F11 P03951 Coagulation factor XI ↓CNDP1 Q96KN2 Beta-Ala-His dipeptidase ↓ BCHE P06276 Cholinesterase ↓APCS P02743 Serum amyloid P-component ↓ APOH P02749 Beta-2-glycoprotein1 ↓ AZGP1 P25311 Zinc-alpha-2-glycoprotein ↓ C4A Q5JQM8 Complement C4-A↓ C6 P13671 Complement component C6 ↓ C8A P07357 Complement component C8alpha ↓ chain C8B P07358 Complement component C8 beta ↓ chain C8G P07360Complement component C8 ↓ gamma chain HPX P02790 Hemopexin ↓ IGFALSP35858 Insulin-like growth factor-binding ↓ protein complex acid labilesubunit ITIH1 P19827 Inter-alpha-trypsin inhibitor heavy ↓ chain H1ITIH2 P19823 Inter-alpha-trypsin inhibitor heavy ↓ chain H2 ITIH4 Q14624Inter-alpha-trypsin inhibitor heavy ↓ chain H4 PRG4 Q92954 Proteoglycan4 ↓ RBP4 P02753 Retinol-binding protein 4 ↓ SERPINC1 Q9UC78Antithrombin-III ↓ SERPIND1 P05546 Heparin cofactor 2 ↓ TTR P02766Transthyretin ↓ APOC3 P02656 Apolipoprotein C-III ↓ F10 P00742Coagulation factor X ↓ SERPINA4 P29622 Kallistatin ↓ VTN P04004Vitronectin ↑ LUM P51884 Lumican ↑ SERPING1 Q96FE0 Plasma protease C1inhibitor ↑ BTD P43251 Biotinidase ↓ PLG P00747 Plasminogen ↓ PROCP04070 Vitamin K-dependent protein C ↓ CPN1 P15169 Carboxypeptidase Ncatalytic chain ↓ CPB2 Q96IY4 Carboxypeptidase B2 ↓ GC P02774 VitaminD-binding protein ↓ GP5 P40197 Platelet glycoprotein V ↓ C2 P06681Complement C2 ↓ F12 P00748 Coagulation factor XII ↓ ANPEP P15144Aminopeptidase N ↑ CP P00450 Ceruloplasmin ↑ TGFBI Q53XM1 Transforminggrowth factor-beta- ↑ induced protein ig-h3 FCGBP Q9Y6R7 IgGFc-bindingprotein ↑ SERPINA7 P05543 Thyroxine-binding globulin ↓ PVR P15151Poliovirus receptor ↑ AGT P01019 Angiotensinogen ↓ H6PD O95479 GDH/6PGLendoplasmic ↓ bifunctional protein PROCR Q9UNN8 Endothelial protein Creceptor ↑ VASN Q6EMK4 Vasorin ↑ — Ig lambda chain V-I region NEWM ↑APOC1 P02654 Apolipoprotein C-I ↑ ICAM1 P05362 Intercellular adhesionmolecule 1 ↑ CSF1R P07333 Macrophage colony-stimulating ↑ factor 1receptor DBH P09172 Dopamine beta-hydroxylase ↑ COL6A3 P12111 Collagenalpha-3(VI) chain ↑ COMP P49747 Cartilage oligomeric matrix protein ↑CD163 Q86VB7 Scavenger receptor cysteine-rich ↑ type 1 protein M130LYVE1 Q9Y5Y7 Lymphatic vessel endothelial ↑ hyaluronic acid receptor 1LCAT P04180 Phosphatidylcholine-sterol ↓ acyltransferase CPN2 P22792Carboxypeptidase N subunit 2 ↓ PROZ P22891 Vitamin K-dependent protein Z↓ MASP1 P48740 Mannan-binding lectin serine ↓ protease 1 SEPP1 P49908Selenoprotein P ↓ RARRES2 Q99969 Retinoic acid receptor responder ↓protein 2

Table 6 lists the liver fibrosis-related proteins with differentialexpression only in fast-progressing (FP) post-transplant patients oronly in slow-progressing (SP) post-transplant patients relative tocontrols representing protein expression in a patient withnon-progressing liver fibrosis that also show differential expression inthe same direction in non-transplant Stage 4-6 Liver Fibrosis subjectsrelative to Controls representing protein expression in a non-transplantStage 0-1 Liver Fibrosis subject. Excluded from Table 6 are twoproteins, F12 and Serpina7, which showed differential expression but inthe opposite direction from the differential expression detected in SPor FP post-transplant subjects. Thus, for 90% (18 out of 20 proteins) ofproteins with differential expression only in FP or SP that also showeddifferential expression in non-transplant patients, the differentialexpression was in the same direction as was detected for FP or SPpost-transplant patients. 10 of the proteins in Table 6 showdifferential expression only in FP post-transplant patients, and 8 ofthe proteins in Table 6 show differential expression only in SPpost-transplant patients.

TABLE 6 Liver Fibrosis Related Proteins with Differential Expression inonly Fast-Progressing (FP) Post-Transplant Patients or onlySlow-Progressing (SP) Post-Transplant patients relative to Controlsrepresenting protein expression in a patient with Non-Progressing LiverFibrosis that show Differential Expression in the same direction inNon-Transplant Stage 4-6 Liver Fibrosis subjects relative to Controlsrepresenting protein expression in a Non-Transplant Stage 0-1 LiverFibrosis subject. A. Proteins with Differential Expression only in SPPost-Transplant Subjects that have co-directional DifferentialExpression in Non-Transplant Stage 4-6 Subjects SERPING1 Q96FE0 Plasmaprotease C1 ↑ inhibitor BTD P43251 Biotinidase ↓ PLG P00747 Plasminogen↓ PROC P04070 Vitamin K-dependent ↓ protein C CPN1 P15169Carboxypeptidase N ↓ catalytic chain CPB2 Q96IY4 Carboxypeptidase B2 ↓GC P02774 Vitamin D-binding ↓ protein GP5 P40197 Platelet glycoprotein V↓ B. Proteins with Differential Expression only in FP Post-TransplantSubjects that have co-directional Differential Expression inNon-Transplant Stage 4-6 Subjects C2 P06681 Complement C2 ↓ ANPEP P15144Aminopeptidase N ↑ CP P00450 Ceruloplasmin ↑ TGFBI Q53XM1 Transforminggrowth ↑ factor-beta-induced protein ig- h3 FCGBP Q9Y6R7 IgGFc-bindingprotein ↑ PVR P15151 Poliovirus receptor ↑ AGT P01019 Angiotensinogen ↓H6PD O95479 GDH/6PGL endoplasmic ↓ bifunctional protein PROCR Q9UNN8Endothelial protein C ↑ receptor VASN Q6EMK4 Vasorin ↑

In a specific example, expression of two or more of the liverfibrosis-related proteins (or corresponding peptides or nucleic acidmolecules) labeled under “Proteins with Differential Expression only inFP Post-Transplant Subjects that have co-directional DifferentialExpression in Non-Transplant Stage 4-6 Subjects” in Table 6 can be usedto diagnose or prognose fast-progressing liver fibrosis in a subject,such as by detecting expression of at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10 suchproteins (or corresponding peptides or nucleic acid molecules) in Table6. For example, if at least two of the liver fibrosis-related moleculesin Table 6 (such as at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, or all 10 labeled under “Proteins withDifferential Expression only in FP Post-Transplant Subjects that haveco-directional Differential Expression in Non-Transplant Stage 4-6Subjects”) show differential expression in the test sample, relative toa control (such as an amount of protein expression expected in a subjector population of subjects having non-progressing liver fibrosis), thisresults in a diagnosis of fast-progressing liver fibrosis, or aprognosis that the subject will develop a fast-progressing liverfibrosis in the future.

In a specific example, expression of two or more of the liverfibrosis-related proteins (or corresponding peptides or nucleic acidmolecules) labeled under “Proteins with Differential Expression only inSP Post-Transplant Subjects that have co-directional DifferentialExpression in Non-Transplant Stage 4-6 Subjects” in Table 6 can be usedto diagnose or prognose slow-progressing liver fibrosis in a subject,such as by detecting expression of at least 3, at least 4, at least 5,at least 6, at least 7, or all 8 such proteins (or correspondingpeptides or nucleic acid molecules) in Table 6. For example, if at leasttwo of the liver fibrosis-related molecules in Table 6 (such as at least3, at least 4, at least 5, at least 6, at least 7, or all 8 labeledunder “Proteins with Differential Expression only in SP Post-TransplantSubjects that have co-directional Differential Expression inNon-Transplant Stage 4-6 Subjects”) show differential expression in thetest sample, relative to a control (such as an amount of proteinexpression expected in a subject or population of subjects havingnon-progressing liver fibrosis), this results in a diagnosis ofslow-progressing liver fibrosis, or a prognosis that the subject willdevelop a slow-progressing liver fibrosis in the future.

IV. Progression of Liver Fibrosis

Rates of liver fibrosis progression can be described as fast, slow ornon-progressing. In general, fast-progressing liver fibrosis progressesto an advanced stage (e.g., Stage 3-4 liver fibrosis) over a time periodshorter than expected. Slow-progressing liver fibrosis progresses to anadvanced stage over a time period longer than expected, andnon-progressing liver fibrosis does not progress to an advanced stage ofliver fibrosis over the course of a reasonable monitoring period. Theexpected rate of progression to liver fibrosis may depend upon theunderlying cause. For example, in subjects having undergone livertransplantation, subjects may be categorized as having non-progressingliver fibrosis if no or mild return of fibrosis occurs over a timeperiod of 2-4 years post-liver transplant. Subjects with slowprogressing liver fibrosis may develop stage 3-4 fibrosis at three tofour years post-transplant. Finally, subjects with fast progressingliver fibrosis may develop Stage 3-4 liver fibrosis within two yearspost-transplant.

Progression to liver fibrosis and advanced liver fibrosis may occur on adifferent schedule for subjects having other conditions causing liverfibrosis such as HCV infection (in a non-transplant subject), HBVinfection, non-alcoholic fatty liver disease (NAFLD), Wilson's disease,alpha-1-antitrypsin deficiency, hemochromatosis, primary biliarycirrhosis, primary sclerosing cholangitis, and autoimmune hepatitis. Forexample, for a non-transplant subject infected with HCV, the expectationmay be set according to the individual's age at the time of infectionwith HCV based on Poynard's fibrosis progression model (Poynard et al.,2001. J. Hepatol. 34: 730-9). For example, according to the Poynardmodel, a normal rate of progression to liver fibrosis in an individualyounger than 20 years of age is 40 years. On the other hand, individualswho are infected at the age of 40 or older will develop liver fibrosisfollowing 10-20 years from the time of infection. For a patient infectedwith HCV, this expectation may be set according to the Poynard fibrosisprogression model discussed above. Accordingly, fast progressing liverfibrosis for a subject infected with HCV may refer to the development ofliver fibrosis (e.g., stage 3 or higher) within a time period which isat least 5 years shorter than expected for a patient. Likewise, slowprogressing liver fibrosis may refer to fibrosis which occurs over atime period which is at least 5 years longer than expected for asubject. Non-progressing liver fibrosis may refer to fibrosis whichfails to develop or only manifests as mild fibrosis (e.g., stage 2 orlower) within a reasonable monitoring period. Based on the observationthat fast-progression and slow-progression to fibrosis occurs over atime period that may depend on liver fibrosis cause, in variousembodiments, fast-progression indicates progression to moderate oradvanced liver fibrosis within less than 2 years, less than 3 years,less than 5 years, less than 10 years, less than 15 years, less than 20years, less than 25 years, less than 30 years, or less than 40 yearsfrom a causative event or onset of a causative condition, such as 1 to 5years, 1 to 10 years, or 2 to 20 years. Likewise, in variousembodiments, slow-progressing liver fibrosis indicates progression tomoderate or advanced liver fibrosis more than 2 years, more than 3years, more than 5 years, more than 10 years, more than 15 years, morethan 20 years, more than 25 years, more than 30 years, or more than 40years after a causative event or onset of a causative condition, such as2 to 5 years, 2 to 10 years, or 5 to 20 years.

The median estimated duration from the HCV infection date to theappearance of cirrhosis (end-stage liver fibrosis) is 30 years andapproximately 33% of the patients progress to cirrhosis in less than 20years (Poynard et al., 1997; Lancet. 349: 825-32). This indicatessubstantial variation in fibrosis progression rate and suggests thatseveral host factors (e.g., factors related to the infected individualand not HCV itself), contribute to fibrosis progression. For example,older age, male gender, alcohol intake and immunosuppressant therapy canbe associated with a less favorable outcome in terms of liver fibrosis[Poynard, supra]. Additionally, genetic factors can determine thenatural history of liver diseases and the progression of liver fibrosis.These include genetic polymorphisms in genes encoding immuno-regulatoryproteins, proinflammatory cytokines, and fibrogenic factors (Bataller etal., 2003. Hepatology. 37:493-503).

V. Detection of Liver Fibrosis-Related Nucleic Acid and ProteinMolecules

The samples obtained from the subject (for example a serum or plasmasample) can contain altered levels of one or more nucleic acids orproteins associated with liver fibrosis, such as those listed in any ofTables 1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544.Relative changes in expression or detected amounts can be detected todetermine if a subject is predisposed to developing liver fibrosis, orhas liver fibrosis. Although much of the disclosure focuses on detectionof liver fibrosis-related proteins, one skilled in the art willappreciate that corresponding nucleic acid molecules (such as RNA, mRNA,DNA or cDNA) can be detected as a means to determine if expression ofliver fibrosis-related molecules is altered. The present disclosure isnot limited to particular methods of detecting proteins or nucleic acidmolecules. Any method of detecting a nucleic acid molecule or proteincan be used, such as physical or functional assays.

For example, the level of gene expression can be measured and quantifiedutilizing methods well known in the art, such as Northern-Blots, RNaseprotection assays, nucleic acid or antibody probe arrays, quantitativePCR (such as TaqMan assays), dot blot assays, in-situ hybridization, orcombinations thereof. Using known techniques, a nucleic acid moleculemay be first amplified prior to detection. In various embodiments,amplification of a nucleic acid or detection of a nucleic acid may beperformed using a manual or automated technique or instrument. Inaddition, proteins can be detected or measured using immunologicalmethods (such as antibody arrays [for example a lateral flow device,such as a point-of-care device, or an ELISA] or immunohistochemistrymethods), quantitative spectroscopic methods (for example massspectrometry, such as liquid chromatography ion mobility massspectrometry (LC-IMS-MS), surface-enhanced laser desorption/ionization(SELDI)-based mass spectrometry or multiple reaction monitoring (MRM)based tandem mass spectrometry), or combinations thereof. In variousembodiments, the expression level of a whole protein including the levelof differential expression versus a control may be determined bydetecting peptide fragments of the whole protein. In various embodimentsdetection of protein or fragment thereof may be performed using a manualor automated technique or instrument.

Methods for labeling nucleic acid molecules and proteins, as well asantibodies, so that they can be detected are well known. Examples ofsuch labels include non-radiolabels and radiolabels. Non-radiolabelsinclude, but are not limited to enzymes, chemiluminescent compounds,fluorophores, metal complexes, haptens, colorimetric agents, dyes, orcombinations thereof. Radiolabels include, but are not limited to, ¹²⁵Iand ³⁵S. Radioactive and fluorescent labeling methods, as well as othermethods known in the art, are suitable for use with the presentdisclosure. In one example, the primers used to amplify the subject'snucleic acids are labeled (such as with biotin, a radiolabel, or afluorophore), thereby permitting detection of the amplicons. In anotherexample, the amplified nucleic acid samples are end-labeled to formlabeled amplified material. For example, amplified nucleic acidmolecules can be labeled by including labeled nucleotides in theamplification reactions. In another example, nucleic acid moleculesobtained from a subject are labeled, and applied to an array containingoligonucleotides. In a particular example, proteins obtained from asubject are labeled and subsequently analyzed, for example by applyingthem to an array.

In one example, a biological sample from the subject is assayed for anincrease or decrease in expression of liver fibrosis related molecules,such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55,56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73,74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107,108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121,122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135,136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149,150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177,178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191,192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219,220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233,234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247,248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261,262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275,276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289,290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303,304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317,318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331,332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345,346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359,360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373,374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387,388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401,402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415,416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443,444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457,458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485,486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499,500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513,514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527,528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541,542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555,556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569,570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583,584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597,598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611,612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625,626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639,640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653,654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667,668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681,682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695,696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707, 708, 709,710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721, 722, 723,724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735, 736, 737,738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749, 750, 751,752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763, 764, 765,766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779,780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793,794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807,808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821,822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835,836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849,850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863,864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877,878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891,892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905,906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919,920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933,934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947,948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961,962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975,976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989,990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002,1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014,1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026,1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038,1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050,1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062,1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074,1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086,1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098,1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110,1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122,1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134,1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146,1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158,1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170,1171, 1172, 1173, 1174, 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2189, 2190,2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201, 2202,2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213, 2214,2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225, 2226,2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237, 2238,2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249, 2250,2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261, 2262,2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273, 2274,2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285, 2286,2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297, 2298,2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309, 2310,2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321, 2322,2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333, 2334,2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345, 2346,2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357, 2358,2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369, 2370,2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381, 2382,2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393, 2394,2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405, 2406,2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417, 2418,2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429, 2430,2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441, 2442,2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453, 2454,2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465, 2466,2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477, 2478,2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489, 2490,2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501, 2502,2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513, 2514,2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525, 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2694,2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705, 2706,2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716, 2717, 2718,2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728, 2729, 2730,2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740, 2741, 2742,2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753, 2754,2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765, 2766,2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776, 2777, 2778,2779, 2780, 2781, 2782, 2783, 2784, 2785, 2786, 2787, 2788, 2789, 2790,2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801, 2802,2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813, 2814,2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825, 2826,2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836, 2837, 2838,2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848, 2849, 2850,2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860, 2861, 2862,2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872, 2873, 2874,2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 2885, 2886,2887, 2888, 2889, 2890, 2891, 2892, 2893, 2894, 2895, 2896, 2897, 2898,2899, 2900, 2901, 2902, 2903, 2904, 2905, 2906, 2907, 2908, 2909, 2910,2911, 2912, 2913, 2914, 2915, 2916, 2917, 2918, 2919, 2920, 2921, 2922,2923, 2924, 2925, 2926, 2927, 2928, 2929, 2930, 2931, 2932, 2933, 2934,2935, 2936, 2937, 2938, 2939, 2940, 2941, 2942, 2943, 2944, 2945, 2946,2947, 2948, 2949, 2950, 2951, 2952, 2953, 2954, 2955, 2956, 2957, 2958,2959, 2960, 2961, 2962, 2963, 2964, 2965, 2966, 2967, 2968, 2969, 2970,2971, 2972, 2973, 2974, 2975, 2976, 2977, 2978, 2979, 2980, 2981, 2982,2983, 2984, 2985, 2986, 2987, 2988, 2989, 2990, 2991, 2992, 2993, 2994,2995, 2996, 2997, 2998, 2999, 3000, 3001, 3002, 3003, 3004, 3005, 3006,3007, 3008, 3009, 3010, 3011, 3012, 3013, 3014, 3015, 3016, 3017, 3018,3019, 3020, 3021, 3022, 3023, 3024, 3025, 3026, 3027, 3028, 3029, 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3198,3199, 3200, 3201, 3202, 3203, 3204, 3205, 3206, 3207, 3208, 3209, 3210,3211, 3212, 3213, 3214, 3215, 3216, 3217, 3218, 3219, 3220, 3221, 3222,3223, 3224, 3225, 3226, 3227, 3228, 3229, 3230, 3231, 3232, 3233, 3234,3235, 3236, 3237, 3238, 3239, 3240, 3241, 3242, 3243, 3244, 3245, 3246,3247, 3248, 3249, 3250, 3251, 3252, 3253, 3254, 3255, 3256, 3257, 3258,3259, 3260, 3261, 3262, 3263, 3264, 3265, 3266, 3267, 3268, 3269, 3270,3271, 3272, 3273, 3274, 3275, 3276, 3277, 3278, 3279, 3280, 3281, 3282,3283, 3284, 3285, 3286, 3287, 3288, 3289, 3290, 3291, 3292, 3293, 3294,3295, 3296, 3297, 3298, 3299, 3300, 3301, 3302, 3303, 3304, 3305, 3306,3307, 3308, 3309, 3310, 3311, 3312, 3313, 3314, 3315, 3316, 3317, 3318,3319, 3320, 3321, 3322, 3323, 3324, 3325, 3326, 3327, 3328, 3329, 3330,3331, 3332, 3333, 3334, 3335, 3336, 3337, 3338, 3339, 3340, 3341, 3342,3343, 3344, 3345, 3346, 3347, 3348, 3349, 3350, 3351, 3352, 3353, 3354,3355, 3356, 3357, 3358, 3359, 3360, 3361, 3362, 3363, 3364, 3365, 3366,3367, 3368, 3369, 3370, 3371, 3372, 3373, 3374, 3375, 3376, 3377, 3378,3379, 3380, 3381, 3382, 3383, 3384, 3385, 3386, 3387, 3388, 3389, 3390,3391, 3392, 3393, 3394, 3395, 3396, 3397, 3398, 3399, 3400, 3401, 3402,3403, 3404, 3405, 3406, 3407, 3408, 3409, 3410, 3411, 3412, 3413, 3414,3415, 3416, 3417, 3418, 3419, 3420, 3421, 3422, 3423, 3424, 3425, 3426,3427, 3428, 3429, 3430, 3431, 3432, 3433, 3434, 3435, 3436, 3437, 3438,3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450,3451, 3452, 3453, 3454, 3455, 3456, 3457, 3458, 3459, 3460, 3461, 3462,3463, 3464, 3465, 3466, 3467, 3468, 3469, 3470, 3471, 3472, 3473, 3474,3475, 3476, 3477, 3478, 3479, 3480, 3481, 3482, 3483, 3484, 3485, 3486,3487, 3488, 3489, 3490, 3491, 3492, 3493, 3494, 3495, 3496, 3497, 3498,3499, 3500, 3501, 3502, 3503, 3504, 3505, 3506, 3507, 3508, 3509, 3510,3511, 3512, 3513, 3514, 3515, 3516, 3517, 3518, 3519, 3520, 3521, 3522,3523, 3524, 3525, 3526, 3527, 3528, 3529, 3530, 3531, 3532, 3533, 3534,3535, 3536, 3537, 3538, 3539, 3540, 3541, 3542, 3543, 3544, 3545, 3546,3547, 3548, 3549, 3550, 3551, 3552, 3553, 3554, 3555, 3556, 3557, 3558,3559, 3560, 3561, 3562, 3563, 3564, 3565, 3566, 3567, 3568, 3569, 3570,3571, 3572, 3573, 3574, 3575, 3576, 3577, 3578, 3579, 3580, 3581, 3582,3583, 3584, 3585, 3586, 3587, 3588, 3589, 3590, 3591, 3592, 3593, 3594,3595, 3596, 3597, 3598, 3599, 3600, 3601, 3602, 3603, 3604, 3605, 3606,3607, 3608, 3609, 3610, 3611, 3612, 3613, 3614, 3615, 3616, 3617, 3618,3619, 3620, 3621, 3622, 3623, 3624, 3625, 3626, 3627, 3628, 3629, 3630,3631, 3632, 3633, 3634, 3635, 3636, 3637, 3638, 3639, 3640, 3641, 3642,3643, 3644, 3645, 3646, 3647, 3648, 3649, 3650, 3651, 3652, 3653, 3654,3655, 3656, 3657, 3658, 3659, 3660, 3661, 3662, 3663, 3664, 3665, 3666,3667, 3668, 3669, 3670, 3671, 3672, 3673, 3674, 3675, 3676, 3677, 3678,3679, 3680, 3681, 3682, 3683, 3684, 3685, 3686, 3687, 3688, 3689, 3690,3691, 3692, 3693, 3694, 3695, 3696, 3697, 3698, 3699, 3700, 3701, 3702,3703, 3704, 3705, 3706, 3707, 3708, 3709, 3710, 3711, 3712, 3713, 3714,3715, 3716, 3717, 3718, 3719, 3720, 3721, 3722, 3723, 3724, 3725, 3726,3727, 3728, 3729, 3730, 3731, 3732, 3733, 3734, 3735, 3736, 3737, 3738,3739, 3740, 3741, 3742, 3743, 3744, 3745, 3746, 3747, 3748, 3749, 3750,3751, 3752, 3753, 3754, 3755, 3756, 3757, 3758, 3759, 3760, 3761, 3762,3763, 3764, 3765, 3766, 3767, 3768, 3769, 3770, 3771, 3772, 3773, 3774,3775, 3776, 3777, 3778, 3779, 3780, 3781, 3782, 3783, 3784, 3785, 3786,3787, 3788, 3789, 3790, 3791, 3792, 3793, 3794, 3795, 3796, 3797, 3798,3799, 3800, 3801, 3802, 3803, 3804, 3805, 3806, 3807, 3808, 3809, 3810,3811, 3812, 3813, 3814, 3815, 3816, 3817, 3818, 3819, 3820, 3821, 3822,3823, 3824, 3825, 3826, 3827, 3828, 3829, 3830, 3831, 3832, 3833, 3834,3835, 3836, 3837, 3838, 3839, 3840, 3841, 3842, 3843, 3844, 3845, 3846,3847, 3848, 3849, 3850, 3851, 3852, 3853, 3854, 3855, 3856, 3857, 3858,3859, 3860, 3861, 3862, 3863, 3864, 3865, 3866, 3867, 3868, 3869, 3870,3871, 3872, 3873, 3874, 3875, 3876, 3877, 3878, 3879, 3880, 3881, 3882,3883, 3884, 3885, 3886, 3887, 3888, 3889, 3890, 3891, 3892, 3893, 3894,3895, 3896, 3897, 3898, 3899, 3900, 3901, 3902, 3903, 3904, 3905, 3906,3907, 3908, 3909, 3910, 3911, 3912, 3913, 3914, 3915, 3916, 3917, 3918,3919, 3920, 3921, 3922, 3923, 3924, 3925, 3926, 3927, 3928, 3929, 3930,3931, 3932, 3933, 3934, 3935, 3936, 3937, 3938, 3939, 3940, 3941, 3942,3943, 3944, 3945, 3946, 3947, 3948, 3949, 3950, 3951, 3952, 3953, 3954,3955, 3956, 3957, 3958, 3959, 3960, 3961, 3962, 3963, 3964, 3965, 3966,3967, 3968, 3969, 3970, 3971, 3972, 3973, 3974, 3975, 3976, 3977, 3978,3979, 3980, 3981, 3982, 3983, 3984, 3985, 3986, 3987, 3988, 3989, 3990,3991, 3992, 3993, 3994, 3995, 3996, 3997, 3998, 3999, 4000, 4001, 4002,4003, 4004, 4005, 4006, 4007, 4008, 4009, 4010, 4011, 4012, 4013, 4014,4015, 4016, 4017, 4018, 4019, 4020, 4021, 4022, 4023, 4024, 4025, 4026,4027, 4028, 4029, 4030, 4031, 4032, 4033, 4034, 4035, 4036, 4037, 4038,4039, 4040, 4041, 4042, 4043, 4044, 4045, 4046, 4047, 4048, 4049, 4050,4051, 4052, 4053, 4054, 4055, 4056, 4057, 4058, 4059, 4060, 4061, 4062,4063, 4064, 4065, 4066, 4067, 4068, 4069, 4070, 4071, 4072, 4073, 4074,4075, 4076, 4077, 4078, 4079, 4080, 4081, 4082, 4083, 4084, 4085, 4086,4087, 4088, 4089, 4090, 4091, 4092, 4093, 4094, 4095, 4096, 4097, 4098,4099, 4100, 4101, 4102, 4103, 4104, 4105, 4106, 4107, 4108, 4109, 4110,4111, 4112, 4113, 4114, 4115, 4116, 4117, 4118, 4119, 4120, 4121, 4122,4123, 4124, 4125, 4126, 4127, 4128, 4129, 4130, 4131, 4132, 4133, 4134,4135, 4136, 4137, 4138, 4139, 4140, 4141, 4142, 4143, 4144, 4145, 4146,4147, 4148, 4149, 4150, 4151, 4152, 4153, 4154, 4155, 4156, 4157, 4158,4159, 4160, 4161, 4162, 4163, 4164, 4165, 4166, 4167, 4168, 4169, 4170,4171, 4172, 4173, 4174, 4175, 4176, 4177, 4178, 4179, 4180, 4181, 4182,4183, 4184, 4185, 4186, 4187, 4188, 4189, 4190, 4191, 4192, 4193, 4194,4195, 4196, 4197, 4198, 4199, 4200, 4201, 4202, 4203, 4204, 4205, 4206,4207, 4208, 4209, 4210, 4211, 4212, 4213, 4214, 4215, 4216, 4217, 4218,4219, 4220, 4221, 4222, 4223, 4224, 4225, 4226, 4227, 4228, 4229, 4230,4231, 4232, 4233, 4234, 4235, 4236, 4237, 4238, 4239, 4240, 4241, 4242,4243, 4244, 4245, 4246, 4247, 4248, 4249, 4250, 4251, 4252, 4253, 4254,4255, 4256, 4257, 4258, 4259, 4260, 4261, 4262, 4263, 4264, 4265, 4266,4267, 4268, 4269, 4270, 4271, 4272, 4273, 4274, 4275, 4276, 4277, 4278,4279, 4280, 4281, 4282, 4283, 4284, 4285, 4286, 4287, 4288, 4289, 4290,4291, 4292, 4293, 4294, 4295, 4296, 4297, 4298, 4299, 4300, 4301, 4302,4303, 4304, 4305, 4306, 4307, 4308, 4309, 4310, 4311, 4312, 4313, 4314,4315, 4316, 4317, 4318, 4319, 4320, 4321, 4322, 4323, 4324, 4325, 4326,4327, 4328, 4329, 4330, 4331, 4332, 4333, 4334, 4335, 4336, 4337, 4338,4339, 4340, 4341, 4342, 4343, 4344, 4345, 4346, 4347, 4348, 4349, 4350,4351, 4352, 4353, 4354, 4355, 4356, 4357, 4358, 4359, 4360, 4361, 4362,4363, 4364, 4365, 4366, 4367, 4368, 4369, 4370, 4371, 4372, 4373, 4374,4375, 4376, 4377, 4378, 4379, 4380, 4381, 4382, 4383, 4384, 4385, 4386,4387, 4388, 4389, 4390, 4391, 4392, 4393, 4394, 4395, 4396, 4397, 4398,4399, 4400, 4401, 4402, 4403, 4404, 4405, 4406, 4407, 4408, 4409, 4410,4411, 4412, 4413, 4414, 4415, 4416, 4417, 4418, 4419, 4420, 4421, 4422,4423, 4424, 4425, 4426, 4427, 4428, 4429, 4430, 4431, 4432, 4433, 4434,4435, 4436, 4437, 4438, 4439, 4440, 4441, 4442, 4443, 4444, 4445, 4446,4447, 4448, 4449, 4450, 4451, 4452, 4453, 4454, 4455, 4456, 4457, 4458,4459, 4460, 4461, 4462, 4463, 4464, 4465, 4466, 4467, 4468, 4469, 4470,4471, 4472, 4473, 4474, 4475, 4476, 4477, 4478, 4479, 4480, 4481, 4482,4483, 4484, 4485, 4486, 4487, 4488, 4489, 4490, 4491, 4492, 4493, 4494,4495, 4496, 4497, 4498, 4499, 4500, 4501, 4502, 4503, 4504, 4505, 4506,4507, 4508, 4509, 4510, 4511, 4512, 4513, 4514, 4515, 4516, 4517, 4518,4519, 4520, 4521, 4522, 4523, 4524, 4525, 4526, 4527, 4528, 4529, 4530,4531, 4532, 4533, 4534, 4535, 4536, 4537, 4538, 4539, 4540, 4541, 4542,4543, 4544, 4545, 4546, 4547, 4548, 4549, 4550, 4551, 4552, 4553, 4554,4555, 4556, 4557, 4558, 4559, 4560, 4561, 4562, 4563, 4564, 4565, 4566,4567, 4568, 4569, 4570, 4571, 4572, 4573, 4574, 4575, 4576, 4577, 4578,4579, 4580, 4581, 4582, 4583, 4584, 4585, 4586, 4587, 4588, 4589, 4590,4591, 4592, 4593, 4594, 4595, 4596, 4597, 4598, 4599, 4600, 4601, 4602,4603, 4604, 4605, 4606, 4607, 4608, 4609, 4610, 4611, 4612, 4613, 4614,4615, 4616, 4617, 4618, 4619, 4620, 4621, 4622, 4623, 4624, 4625, 4626,4627, 4628, 4629, 4630, 4631, 4632, 4633, 4634, 4635, 4636, 4637, 4638,4639, 4640, 4641, 4642, 4643, 4644, 4645, 4646, 4647, 4648, 4649, 4650,4651, 4652, 4653, 4654, 4655, 4656, 4657, 4658, 4659, 4660, 4661, 4662,4663, 4664, 4665, 4666, 4667, 4668, 4669, 4670, 4671, 4672, 4673, 4674,4675, 4676, 4677, 4678, 4679, 4680, 4681, 4682, 4683, 4684, 4685, 4686,4687, 4688, 4689, 4690, 4691, 4692, 4693, 4694, 4695, 4696, 4697, 4698,4699, 4700, 4701, 4702, 4703, 4704, 4705, 4706, 4707, 4708, 4709, 4710,4711, 4712, 4713, 4714, 4715, 4716, 4717, 4718, 4719, 4720, 4721, 4722,4723, 4724, 4725, 4726, 4727, 4728, 4729, 4730, 4731, 4732, 4733, 4734,4735, 4736, 4737, 4738, 4739, 4740, 4741, 4742, 4743, 4744, 4745, 4746,4747, 4748, 4749, 4750, 4751, 4752, 4753, 4754, 4755, 4756, 4757, 4758,4759, 4760, 4761, 4762, 4763, 4764, 4765, 4766, 4767, 4768, 4769, 4770,4771, 4772, 4773, 4774, 4775, 4776, 4777, 4778, 4779, 4780, 4781, 4782,4783, 4784, 4785, 4786, 4787, 4788, 4789, 4790, 4791, 4792, 4793, 4794,4795, 4796, 4797, 4798, 4799, 4800, 4801, 4802, 4803, 4804, 4805, 4806,4807, 4808, 4809, 4810, 4811, 4812, 4813, 4814, 4815, 4816, 4817, 4818,4819, 4820, 4821, 4822, 4823, 4824, 4825, 4826, 4827, 4828, 4829, 4830,4831, 4832, 4833, 4834, 4835, 4836, 4837, 4838, 4839, 4840, 4841, 4842,4843, 4844, 4845, 4846, 4847, 4848, 4849, 4850, 4851, 4852, 4853, 4854,4855, 4856, 4857, 4858, 4859, 4860, 4861, 4862, 4863, 4864, 4865, 4866,4867, 4868, 4869, 4870, 4871, 4872, 4873, 4874, 4875, 4876, 4877, 4878,4879, 4880, 4881, 4882, 4883, 4884, 4885, 4886, 4887, 4888, 4889, 4890,4891, 4892, 4893, 4894, 4895, 4896, 4897, 4898, 4899, 4900, 4901, 4902,4903, 4904, 4905, 4906, 4907, 4908, 4909, 4910, 4911, 4912, 4913, 4914,4915, 4916, 4917, 4918, 4919, 4920, 4921, 4922, 4923, 4924, 4925, 4926,4927, 4928, 4929, 4930, 4931, 4932, 4933, 4934, 4935, 4936, 4937, 4938,4939, 4940, 4941, 4942, 4943, 4944, 4945, 4946, 4947, 4948, 4949, 4950,4951, 4952, 4953, 4954, 4955, 4956, 4957, 4958, 4959, 4960, 4961, 4962,4963, 4964, 4965, 4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973, 4974,4975, 4976, 4977, 4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985, 4986,4987, 4988, 4989, 4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997, 4998,4999, 5000, 5001, 5002, 5003, 5004, 5005, 5006, 5007, 5008, 5009, 5010,5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021, 5022,5023, 5024, 5025, 5026, 5027, 5028, 5029, 5030, 5031, 5032, 5033, 5034,5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045, 5046,5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057, 5058,5059, 5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069, 5070,5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5080, 5081, 5082,5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090, 5091, 5092, 5093, 5094,5095, 5096, 5097, 5098, 5099, 5100, 5101, 5102, 5103, 5104, 5105, 5106,5107, 5108, 5109, 5110, 5111, 5112, 5113, 5114, 5115, 5116, 5117, 5118,5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128, 5129, 5130,5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140, 5141, 5142,5143, 5144, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153, 5154,5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165, 5166,5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177, 5178,5179, 5180, 5181, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189, 5190,5191, 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201, 5202,5203, 5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213, 5214,5215, 5216, 5217, 5218, 5219, 5220, 5221, 5222, 5223, 5224, 5225, 5226,5227, 5228, 5229, 5230, 5231, 5232, 5233, 5234, 5235, 5236, 5237, 5238,5239, 5240, 5241, 5242, 5243, 5244, 5245, 5246, 5247, 5248, 5249, 5250,5251, 5252, 5253, 5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261, 5262,5263, 5264, 5265, 5266, 5267, 5268, 5269, 5270, 5271, 5272, 5273, 5274,5275, 5276, 5277, 5278, 5279, 5280, 5281, 5282, 5283, 5284, 5285, 5286,5287, 5288, 5289, 5290, 5291, 5292, 5293, 5294, 5295, 5296, 5297, 5298,5299, 5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309, 5310,5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321, 5322,5323, 5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333, 5334,5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345, 5346,5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357, 5358,5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369, 5370,5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381, 5382,5383, 5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393, 5394,5395, 5396, 5397, 5398, 5399, 5400, 5401, 5402, 5403, 5404, 5405, 5406,5407, 5408, 5409, 5410, 5411, 5412, 5413, 5414, 5415, 5416, 5417, 5418,5419, 5420, 5421, 5422, 5423, 5424, 5425, 5426, 5427, 5428, 5429, 5430,5431, 5432, 5433, 5434, 5435, 5436, 5437, 5438, 5439, 5440, 5441, 5442,5443, 5444, 5445, 5446, 5447, 5448, 5449, 5450, 5451, 5452, 5453, 5454,5455, 5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464, 5465, 5466,5467, 5468, 5469, 5470, 5471, 5472, 5473, 5474, 5475, 5476, 5477, 5478,5479, 5480, 5481, 5482, 5483, 5484, 5485, 5486, 5487, 5488, 5489, 5490,5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5500, 5501, 5502,5503, 5504, 5505, 5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513, 5514,5515, 5516, 5517, 5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525, 5526,5527, 5528, 5529, 5530, 5531, 5532, 5533, 5534, 5535, 5536, 5537, 5538,5539, 5540, 5541, 5542, 5543, or 5544 of the peptides shown in any ofSEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105,106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133,134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147,148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161,162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175,176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189,190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203,204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217,218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231,232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245,246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259,260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273,274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287,288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301,302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329,330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343,344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371,372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385,386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399,400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413,414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427,428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441,442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455,456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469,470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483,484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497,498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511,512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525,526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539,540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553,554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567,568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581,582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595,596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609,610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623,624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637,638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651,652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665,666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679,680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693,694, 695, 696, 697, 698, 699, 700, 701, 702, 703, 704, 705, 706, 707,708, 709, 710, 711, 712, 713, 714, 715, 716, 717, 718, 719, 720, 721,722, 723, 724, 725, 726, 727, 728, 729, 730, 731, 732, 733, 734, 735,736, 737, 738, 739, 740, 741, 742, 743, 744, 745, 746, 747, 748, 749,750, 751, 752, 753, 754, 755, 756, 757, 758, 759, 760, 761, 762, 763,764, 765, 766, 767, 768, 769, 770, 771, 772, 773, 774, 775, 776, 777,778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791,792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805,806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819,820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833,834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847,848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861,862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875,876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889,890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903,904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917,918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931,932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945,946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959,960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973,974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987,988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001,1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013,1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025,1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037,1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049,1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061,1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073,1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085,1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097,1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109,1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121,1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133,1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145,1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157,1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169,1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181,1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193,1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205,1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217,1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229,1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241,1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253,1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265,1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277,1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289,1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301,1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313,1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325,1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337,1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349,1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361,1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373,1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385,1386, 1387, 1388, 1389, 1390, 1391, 1392, 1393, 1394, 1395, 1396, 1397,1398, 1399, 1400, 1401, 1402, 1403, 1404, 1405, 1406, 1407, 1408, 1409,1410, 1411, 1412, 1413, 1414, 1415, 1416, 1417, 1418, 1419, 1420, 1421,1422, 1423, 1424, 1425, 1426, 1427, 1428, 1429, 1430, 1431, 1432, 1433,1434, 1435, 1436, 1437, 1438, 1439, 1440, 1441, 1442, 1443, 1444, 1445,1446, 1447, 1448, 1449, 1450, 1451, 1452, 1453, 1454, 1455, 1456, 1457,1458, 1459, 1460, 1461, 1462, 1463, 1464, 1465, 1466, 1467, 1468, 1469,1470, 1471, 1472, 1473, 1474, 1475, 1476, 1477, 1478, 1479, 1480, 1481,1482, 1483, 1484, 1485, 1486, 1487, 1488, 1489, 1490, 1491, 1492, 1493,1494, 1495, 1496, 1497, 1498, 1499, 1500, 1501, 1502, 1503, 1504, 1505,1506, 1507, 1508, 1509, 1510, 1511, 1512, 1513, 1514, 1515, 1516, 1517,1518, 1519, 1520, 1521, 1522, 1523, 1524, 1525, 1526, 1527, 1528, 1529,1530, 1531, 1532, 1533, 1534, 1535, 1536, 1537, 1538, 1539, 1540, 1541,1542, 1543, 1544, 1545, 1546, 1547, 1548, 1549, 1550, 1551, 1552, 1553,1554, 1555, 1556, 1557, 1558, 1559, 1560, 1561, 1562, 1563, 1564, 1565,1566, 1567, 1568, 1569, 1570, 1571, 1572, 1573, 1574, 1575, 1576, 1577,1578, 1579, 1580, 1581, 1582, 1583, 1584, 1585, 1586, 1587, 1588, 1589,1590, 1591, 1592, 1593, 1594, 1595, 1596, 1597, 1598, 1599, 1600, 1601,1602, 1603, 1604, 1605, 1606, 1607, 1608, 1609, 1610, 1611, 1612, 1613,1614, 1615, 1616, 1617, 1618, 1619, 1620, 1621, 1622, 1623, 1624, 1625,1626, 1627, 1628, 1629, 1630, 1631, 1632, 1633, 1634, 1635, 1636, 1637,1638, 1639, 1640, 1641, 1642, 1643, 1644, 1645, 1646, 1647, 1648, 1649,1650, 1651, 1652, 1653, 1654, 1655, 1656, 1657, 1658, 1659, 1660, 1661,1662, 1663, 1664, 1665, 1666, 1667, 1668, 1669, 1670, 1671, 1672, 1673,1674, 1675, 1676, 1677, 1678, 1679, 1680, 1681, 1682, 1683, 1684, 1685,1686, 1687, 1688, 1689, 1690, 1691, 1692, 1693, 1694, 1695, 1696, 1697,1698, 1699, 1700, 1701, 1702, 1703, 1704, 1705, 1706, 1707, 1708, 1709,1710, 1711, 1712, 1713, 1714, 1715, 1716, 1717, 1718, 1719, 1720, 1721,1722, 1723, 1724, 1725, 1726, 1727, 1728, 1729, 1730, 1731, 1732, 1733,1734, 1735, 1736, 1737, 1738, 1739, 1740, 1741, 1742, 1743, 1744, 1745,1746, 1747, 1748, 1749, 1750, 1751, 1752, 1753, 1754, 1755, 1756, 1757,1758, 1759, 1760, 1761, 1762, 1763, 1764, 1765, 1766, 1767, 1768, 1769,1770, 1771, 1772, 1773, 1774, 1775, 1776, 1777, 1778, 1779, 1780, 1781,1782, 1783, 1784, 1785, 1786, 1787, 1788, 1789, 1790, 1791, 1792, 1793,1794, 1795, 1796, 1797, 1798, 1799, 1800, 1801, 1802, 1803, 1804, 1805,1806, 1807, 1808, 1809, 1810, 1811, 1812, 1813, 1814, 1815, 1816, 1817,1818, 1819, 1820, 1821, 1822, 1823, 1824, 1825, 1826, 1827, 1828, 1829,1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841,1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853,1854, 1855, 1856, 1857, 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865,1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877,1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889,1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 1900, 1901,1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913,1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925,1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937,1938, 1939, 1940, 1941, 1942, 1943, 1944, 1945, 1946, 1947, 1948, 1949,1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961,1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973,1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985,1986, 1987, 1988, 1989, 1990, 1991, 1992, 1993, 1994, 1995, 1996, 1997,1998, 1999, 2000, 2001, 2002, 2003, 2004, 2005, 2006, 2007, 2008, 2009,2010, 2011, 2012, 2013, 2014, 2015, 2016, 2017, 2018, 2019, 2020, 2021,2022, 2023, 2024, 2025, 2026, 2027, 2028, 2029, 2030, 2031, 2032, 2033,2034, 2035, 2036, 2037, 2038, 2039, 2040, 2041, 2042, 2043, 2044, 2045,2046, 2047, 2048, 2049, 2050, 2051, 2052, 2053, 2054, 2055, 2056, 2057,2058, 2059, 2060, 2061, 2062, 2063, 2064, 2065, 2066, 2067, 2068, 2069,2070, 2071, 2072, 2073, 2074, 2075, 2076, 2077, 2078, 2079, 2080, 2081,2082, 2083, 2084, 2085, 2086, 2087, 2088, 2089, 2090, 2091, 2092, 2093,2094, 2095, 2096, 2097, 2098, 2099, 2100, 2101, 2102, 2103, 2104, 2105,2106, 2107, 2108, 2109, 2110, 2111, 2112, 2113, 2114, 2115, 2116, 2117,2118, 2119, 2120, 2121, 2122, 2123, 2124, 2125, 2126, 2127, 2128, 2129,2130, 2131, 2132, 2133, 2134, 2135, 2136, 2137, 2138, 2139, 2140, 2141,2142, 2143, 2144, 2145, 2146, 2147, 2148, 2149, 2150, 2151, 2152, 2153,2154, 2155, 2156, 2157, 2158, 2159, 2160, 2161, 2162, 2163, 2164, 2165,2166, 2167, 2168, 2169, 2170, 2171, 2172, 2173, 2174, 2175, 2176, 2177,2178, 2179, 2180, 2181, 2182, 2183, 2184, 2185, 2186, 2187, 2188, 2189,2190, 2191, 2192, 2193, 2194, 2195, 2196, 2197, 2198, 2199, 2200, 2201,2202, 2203, 2204, 2205, 2206, 2207, 2208, 2209, 2210, 2211, 2212, 2213,2214, 2215, 2216, 2217, 2218, 2219, 2220, 2221, 2222, 2223, 2224, 2225,2226, 2227, 2228, 2229, 2230, 2231, 2232, 2233, 2234, 2235, 2236, 2237,2238, 2239, 2240, 2241, 2242, 2243, 2244, 2245, 2246, 2247, 2248, 2249,2250, 2251, 2252, 2253, 2254, 2255, 2256, 2257, 2258, 2259, 2260, 2261,2262, 2263, 2264, 2265, 2266, 2267, 2268, 2269, 2270, 2271, 2272, 2273,2274, 2275, 2276, 2277, 2278, 2279, 2280, 2281, 2282, 2283, 2284, 2285,2286, 2287, 2288, 2289, 2290, 2291, 2292, 2293, 2294, 2295, 2296, 2297,2298, 2299, 2300, 2301, 2302, 2303, 2304, 2305, 2306, 2307, 2308, 2309,2310, 2311, 2312, 2313, 2314, 2315, 2316, 2317, 2318, 2319, 2320, 2321,2322, 2323, 2324, 2325, 2326, 2327, 2328, 2329, 2330, 2331, 2332, 2333,2334, 2335, 2336, 2337, 2338, 2339, 2340, 2341, 2342, 2343, 2344, 2345,2346, 2347, 2348, 2349, 2350, 2351, 2352, 2353, 2354, 2355, 2356, 2357,2358, 2359, 2360, 2361, 2362, 2363, 2364, 2365, 2366, 2367, 2368, 2369,2370, 2371, 2372, 2373, 2374, 2375, 2376, 2377, 2378, 2379, 2380, 2381,2382, 2383, 2384, 2385, 2386, 2387, 2388, 2389, 2390, 2391, 2392, 2393,2394, 2395, 2396, 2397, 2398, 2399, 2400, 2401, 2402, 2403, 2404, 2405,2406, 2407, 2408, 2409, 2410, 2411, 2412, 2413, 2414, 2415, 2416, 2417,2418, 2419, 2420, 2421, 2422, 2423, 2424, 2425, 2426, 2427, 2428, 2429,2430, 2431, 2432, 2433, 2434, 2435, 2436, 2437, 2438, 2439, 2440, 2441,2442, 2443, 2444, 2445, 2446, 2447, 2448, 2449, 2450, 2451, 2452, 2453,2454, 2455, 2456, 2457, 2458, 2459, 2460, 2461, 2462, 2463, 2464, 2465,2466, 2467, 2468, 2469, 2470, 2471, 2472, 2473, 2474, 2475, 2476, 2477,2478, 2479, 2480, 2481, 2482, 2483, 2484, 2485, 2486, 2487, 2488, 2489,2490, 2491, 2492, 2493, 2494, 2495, 2496, 2497, 2498, 2499, 2500, 2501,2502, 2503, 2504, 2505, 2506, 2507, 2508, 2509, 2510, 2511, 2512, 2513,2514, 2515, 2516, 2517, 2518, 2519, 2520, 2521, 2522, 2523, 2524, 2525,2526, 2527, 2528, 2529, 2530, 2531, 2532, 2533, 2534, 2535, 2536, 2537,2538, 2539, 2540, 2541, 2542, 2543, 2544, 2545, 2546, 2547, 2548, 2549,2550, 2551, 2552, 2553, 2554, 2555, 2556, 2557, 2558, 2559, 2560, 2561,2562, 2563, 2564, 2565, 2566, 2567, 2568, 2569, 2570, 2571, 2572, 2573,2574, 2575, 2576, 2577, 2578, 2579, 2580, 2581, 2582, 2583, 2584, 2585,2586, 2587, 2588, 2589, 2590, 2591, 2592, 2593, 2594, 2595, 2596, 2597,2598, 2599, 2600, 2601, 2602, 2603, 2604, 2605, 2606, 2607, 2608, 2609,2610, 2611, 2612, 2613, 2614, 2615, 2616, 2617, 2618, 2619, 2620, 2621,2622, 2623, 2624, 2625, 2626, 2627, 2628, 2629, 2630, 2631, 2632, 2633,2634, 2635, 2636, 2637, 2638, 2639, 2640, 2641, 2642, 2643, 2644, 2645,2646, 2647, 2648, 2649, 2650, 2651, 2652, 2653, 2654, 2655, 2656, 2657,2658, 2659, 2660, 2661, 2662, 2663, 2664, 2665, 2666, 2667, 2668, 2669,2670, 2671, 2672, 2673, 2674, 2675, 2676, 2677, 2678, 2679, 2680, 2681,2682, 2683, 2684, 2685, 2686, 2687, 2688, 2689, 2690, 2691, 2692, 2693,2694, 2695, 2696, 2697, 2698, 2699, 2700, 2701, 2702, 2703, 2704, 2705,2706, 2707, 2708, 2709, 2710, 2711, 2712, 2713, 2714, 2715, 2716, 2717,2718, 2719, 2720, 2721, 2722, 2723, 2724, 2725, 2726, 2727, 2728, 2729,2730, 2731, 2732, 2733, 2734, 2735, 2736, 2737, 2738, 2739, 2740, 2741,2742, 2743, 2744, 2745, 2746, 2747, 2748, 2749, 2750, 2751, 2752, 2753,2754, 2755, 2756, 2757, 2758, 2759, 2760, 2761, 2762, 2763, 2764, 2765,2766, 2767, 2768, 2769, 2770, 2771, 2772, 2773, 2774, 2775, 2776, 2777,2778, 2779, 2780, 2781, 2782, 2783, 2784, 2785, 2786, 2787, 2788, 2789,2790, 2791, 2792, 2793, 2794, 2795, 2796, 2797, 2798, 2799, 2800, 2801,2802, 2803, 2804, 2805, 2806, 2807, 2808, 2809, 2810, 2811, 2812, 2813,2814, 2815, 2816, 2817, 2818, 2819, 2820, 2821, 2822, 2823, 2824, 2825,2826, 2827, 2828, 2829, 2830, 2831, 2832, 2833, 2834, 2835, 2836, 2837,2838, 2839, 2840, 2841, 2842, 2843, 2844, 2845, 2846, 2847, 2848, 2849,2850, 2851, 2852, 2853, 2854, 2855, 2856, 2857, 2858, 2859, 2860, 2861,2862, 2863, 2864, 2865, 2866, 2867, 2868, 2869, 2870, 2871, 2872, 2873,2874, 2875, 2876, 2877, 2878, 2879, 2880, 2881, 2882, 2883, 2884, 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4901,4902, 4903, 4904, 4905, 4906, 4907, 4908, 4909, 4910, 4911, 4912, 4913,4914, 4915, 4916, 4917, 4918, 4919, 4920, 4921, 4922, 4923, 4924, 4925,4926, 4927, 4928, 4929, 4930, 4931, 4932, 4933, 4934, 4935, 4936, 4937,4938, 4939, 4940, 4941, 4942, 4943, 4944, 4945, 4946, 4947, 4948, 4949,4950, 4951, 4952, 4953, 4954, 4955, 4956, 4957, 4958, 4959, 4960, 4961,4962, 4963, 4964, 4965, 4966, 4967, 4968, 4969, 4970, 4971, 4972, 4973,4974, 4975, 4976, 4977, 4978, 4979, 4980, 4981, 4982, 4983, 4984, 4985,4986, 4987, 4988, 4989, 4990, 4991, 4992, 4993, 4994, 4995, 4996, 4997,4998, 4999, 5000, 5001, 5002, 5003, 5004, 5005, 5006, 5007, 5008, 5009,5010, 5011, 5012, 5013, 5014, 5015, 5016, 5017, 5018, 5019, 5020, 5021,5022, 5023, 5024, 5025, 5026, 5027, 5028, 5029, 5030, 5031, 5032, 5033,5034, 5035, 5036, 5037, 5038, 5039, 5040, 5041, 5042, 5043, 5044, 5045,5046, 5047, 5048, 5049, 5050, 5051, 5052, 5053, 5054, 5055, 5056, 5057,5058, 5059, 5060, 5061, 5062, 5063, 5064, 5065, 5066, 5067, 5068, 5069,5070, 5071, 5072, 5073, 5074, 5075, 5076, 5077, 5078, 5079, 5080, 5081,5082, 5083, 5084, 5085, 5086, 5087, 5088, 5089, 5090, 5091, 5092, 5093,5094, 5095, 5096, 5097, 5098, 5099, 5100, 5101, 5102, 5103, 5104, 5105,5106, 5107, 5108, 5109, 5110, 5111, 5112, 5113, 5114, 5115, 5116, 5117,5118, 5119, 5120, 5121, 5122, 5123, 5124, 5125, 5126, 5127, 5128, 5129,5130, 5131, 5132, 5133, 5134, 5135, 5136, 5137, 5138, 5139, 5140, 5141,5142, 5143, 5144, 5145, 5146, 5147, 5148, 5149, 5150, 5151, 5152, 5153,5154, 5155, 5156, 5157, 5158, 5159, 5160, 5161, 5162, 5163, 5164, 5165,5166, 5167, 5168, 5169, 5170, 5171, 5172, 5173, 5174, 5175, 5176, 5177,5178, 5179, 5180, 5181, 5182, 5183, 5184, 5185, 5186, 5187, 5188, 5189,5190, 5191, 5192, 5193, 5194, 5195, 5196, 5197, 5198, 5199, 5200, 5201,5202, 5203, 5204, 5205, 5206, 5207, 5208, 5209, 5210, 5211, 5212, 5213,5214, 5215, 5216, 5217, 5218, 5219, 5220, 5221, 5222, 5223, 5224, 5225,5226, 5227, 5228, 5229, 5230, 5231, 5232, 5233, 5234, 5235, 5236, 5237,5238, 5239, 5240, 5241, 5242, 5243, 5244, 5245, 5246, 5247, 5248, 5249,5250, 5251, 5252, 5253, 5254, 5255, 5256, 5257, 5258, 5259, 5260, 5261,5262, 5263, 5264, 5265, 5266, 5267, 5268, 5269, 5270, 5271, 5272, 5273,5274, 5275, 5276, 5277, 5278, 5279, 5280, 5281, 5282, 5283, 5284, 5285,5286, 5287, 5288, 5289, 5290, 5291, 5292, 5293, 5294, 5295, 5296, 5297,5298, 5299, 5300, 5301, 5302, 5303, 5304, 5305, 5306, 5307, 5308, 5309,5310, 5311, 5312, 5313, 5314, 5315, 5316, 5317, 5318, 5319, 5320, 5321,5322, 5323, 5324, 5325, 5326, 5327, 5328, 5329, 5330, 5331, 5332, 5333,5334, 5335, 5336, 5337, 5338, 5339, 5340, 5341, 5342, 5343, 5344, 5345,5346, 5347, 5348, 5349, 5350, 5351, 5352, 5353, 5354, 5355, 5356, 5357,5358, 5359, 5360, 5361, 5362, 5363, 5364, 5365, 5366, 5367, 5368, 5369,5370, 5371, 5372, 5373, 5374, 5375, 5376, 5377, 5378, 5379, 5380, 5381,5382, 5383, 5384, 5385, 5386, 5387, 5388, 5389, 5390, 5391, 5392, 5393,5394, 5395, 5396, 5397, 5398, 5399, 5400, 5401, 5402, 5403, 5404, 5405,5406, 5407, 5408, 5409, 5410, 5411, 5412, 5413, 5414, 5415, 5416, 5417,5418, 5419, 5420, 5421, 5422, 5423, 5424, 5425, 5426, 5427, 5428, 5429,5430, 5431, 5432, 5433, 5434, 5435, 5436, 5437, 5438, 5439, 5440, 5441,5442, 5443, 5444, 5445, 5446, 5447, 5448, 5449, 5450, 5451, 5452, 5453,5454, 5455, 5456, 5457, 5458, 5459, 5460, 5461, 5462, 5463, 5464, 5465,5466, 5467, 5468, 5469, 5470, 5471, 5472, 5473, 5474, 5475, 5476, 5477,5478, 5479, 5480, 5481, 5482, 5483, 5484, 5485, 5486, 5487, 5488, 5489,5490, 5491, 5492, 5493, 5494, 5495, 5496, 5497, 5498, 5499, 5500, 5501,5502, 5503, 5504, 5505, 5506, 5507, 5508, 5509, 5510, 5511, 5512, 5513,5514, 5515, 5516, 5517, 5518, 5519, 5520, 5521, 5522, 5523, 5524, 5525,5526, 5527, 5528, 5529, 5530, 5531, 5532, 5533, 5534, 5535, 5536, 5537,5538, 5539, 5540, 5541, 5542, 5543, and 5544.

A. Samples

A biological sample from the subject is assayed for an increase ordecrease in expression of liver fibrosis related molecules, such asthose listed in any of Tables 1, 2, 3, 5, 6 and 12, as well as any ofSEQ ID NOS: 1-5544. Suitable biological samples include those containingDNA or RNA (including cDNA and mRNA) or proteins (including peptidefragments of full-length proteins). Biological samples can be useddirectly, or filtered, denatured, treated with enzymes, concentrated,diluted, or combinations thereof, before the analysis. In some examples,the sample is depleted of high-abundance proteins.

Exemplary samples include blood or fractions thereof, such as a serum orplasma sample. In some examples, the sample is a liver sample, such as abiopsy or fine needle aspirate sample. In a particular example, thesample is a serum or plasma sample that has been immunodepleted. Inanother particular example, the sample is a serum or plasma samplewithout immunodepletion.

B. Nucleic acid Detection

The detection in the biological sample of increased or decreasedexpression of a plurality of liver fibrosis related nucleic acids(nucleic acid sequences corresponding to the liver fibrosis relatedproteins), such as those related to the proteins listed in any of Tables1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544 can beachieved by methods known in the art. For example, increased ordecreased expression of a liver fibrosis related protein can be detectedby measuring the cellular level of mRNA specific for the liver fibrosisrelated protein. mRNA can be measured using techniques well known in theart, including for instance Northern analysis, RT-PCR and mRNA in situhybridization. Details of mRNA analysis procedures can be found, forinstance, in provided examples and in Sambrook et al. (ed.), MolecularCloning: A Laboratory Manual, 2nd ed., vol. 1-3, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989. In addition, genomicDNA and cDNA can be detected using routine methods.

Oligonucleotides specific to liver fibrosis related nucleic acids can bechemically synthesized using commercially available machines. Theseoligonucleotides can then be labeled, for example with radioactiveisotopes (such as ³²P) or with non-radioactive labels such as biotin(Ward and Langer et al., Proc. Natl. Acad. Sci. USA 78:6633-57, 1981) ora fluorophore, and hybridized to individual DNA samples immobilized onmembranes or other solid supports by dot-blot or transfer from gelsafter electrophoresis. These specific sequences are visualized, forexample by methods such as autoradiography or fluorometric (Landegren etal., Science 242:229-37, 1989) or colorimetric reactions (Gebeyehu etal., Nucleic Acids Res. 15:4513-34, 1987), such as SISH or FISH.

Nucleic acid molecules isolated from samples can be amplified usingroutine methods to form nucleic acid amplification products (amplicons,for example by contacting the sample with appropriate primers). In someexamples, the resulting amplicons are detected during amplification. Inother examples, the amplicons are detected following an amplificationreaction. For example, amplicons can then be contacted with one or moreoligonucleotide probes that will hybridize under stringent conditionswith a liver fibrosis related nucleic acid. The nucleic acidamplification products which hybridize with the probe are then detectedand quantified. The oligonucleotide probes or primers can bindspecifically to a nucleic acid molecule that encodes a protein orpeptide listed in any of Tables 1, 2, 3, 5, 6 and 12, as well as any ofSEQ ID NOS: 1-5544.

The nucleic acid molecules obtained from the subject that are associatedwith the liver fibrosis related proteins can be applied to a liverfibrosis detection array under suitable hybridization conditions to forma hybridization complex. For example, the array can includeoligonucleotide probes that can hybridize to liver fibrosis relatednucleic acid molecules in the sample, such as different oligonucleotideprobes on different portions of the array. In particular examples, thenucleic acid molecules in the test sample are labeled, therebypermitting their detection upon hybridization to the oligonucleotideprobe on the array. In one example, a pre-treatment solution of organiccompounds, solutions that include organic compounds, or hot water, canbe applied before hybridization (see U.S. Pat. No. 5,985,567, hereinincorporated by reference).

Hybridization conditions for a given combination of array and targetmaterial can be optimized routinely in an empirical manner close to theT_(m) of the expected duplexes, thereby maximizing the discriminatingpower of the method. Identification of the location in the array, suchas a cell, in which binding occurs, permits a rapid and accurateidentification of sequences associated with liver fibrosis present inthe amplified material (see below).

The hybridization conditions are selected to permit discriminationbetween matched and mismatched oligonucleotides. Hybridizationconditions can be chosen to correspond to those known to be suitable instandard procedures for hybridization to filters and then optimized foruse with the arrays of the disclosure. For example, conditions suitablefor hybridization of one type of target would be adjusted for the use ofother targets for the array. In particular, temperature is controlled tosubstantially eliminate formation of duplexes between sequences otherthan exactly complementary liver fibrosis related nucleic acids. Avariety of known hybridization solvents can be employed, the choicebeing dependent on considerations known to one of skill in the art (seeU.S. Pat. No. 5,981,185).

Once the liver fibrosis related nucleic acid from the subject have beenhybridized with the oligonucleotides present in the liver fibrosisdetection array, the presence of the hybridization complex can beanalyzed, for example by detecting the complexes.

Detecting a hybridized complex in an array of oligonucleotide probes hasbeen previously described (see U.S. Pat. No. 5,985,567). In one example,detection includes detecting one or more labels present on theoligonucleotides, the sequences obtained from the subject, or both. Inparticular examples, developing includes applying a buffer. In oneexample, the buffer is or includes sodium saline citrate, sodium salinephosphate, tetramethylammonium chloride, sodium saline citrate inethylenediaminetetra-acetic, sodium saline citrate in sodium dodecylsulfate, sodium saline phosphate in ethylenediaminetetra-acetic, sodiumsaline phosphate in sodium dodecyl sulfate, tetramethylammonium chloridein ethylenediaminetetra-acetic, tetramethylammonium chloride in sodiumdodecyl sulfate, or combinations thereof. However, other suitable buffersolutions can be used.

Detection can further include treating the hybridized complex with aconjugating solution to effect conjugation or coupling of the hybridizedcomplex with the detection label, and treating the conjugated,hybridized complex with a detection reagent. In one example, theconjugating solution includes streptavidin alkaline phosphatase, avidinalkaline phosphatase, or horseradish peroxidase. Specific, non-limitingexamples of conjugating solutions include streptavidin alkalinephosphatase, avidin alkaline phosphatase, or horseradish peroxidase. Theconjugated, hybridized complex can be treated with a detection reagent.In one example, the detection reagent includes enzyme-labeledfluorescence reagents or calorimetric reagents. In one specificnon-limiting example, the detection reagent is enzyme-labeledfluorescence reagent (ELF) from Molecular Probes, Inc. (Eugene, Oreg.).The hybridized complex can then be placed on a detection device, such asan ultraviolet (UV) transilluminator (manufactured by UVP, Inc. ofUpland, Calif.). The signal is developed and the increased signalintensity can be recorded with a recording device, such as a chargecoupled device (CCD) camera (manufactured by Photometrics, Inc. ofTucson, Ariz.). In particular examples, these steps are not performedwhen fluorophores or radiolabels are used.

In particular examples, the method further includes quantification, forinstance by determining the amount of hybridization or amount ofamplicons produced following amplification (e.g., using PCR).

C. Protein Detection

Protein expression can be detected using any method known in the art,such as by detecting full-length proteins or portions thereof usingantibodies or other specific binding agents such as aptamers oraptazymes, or using other methods such as mass spectrometry. Thedetermination of increased or decreased liver fibrosis related proteinlevels, in comparison to such expression in a control (such as a subjectwho does not have liver fibrosis or has non-progressing liver fibrosis),is an alternative or supplemental approach to the direct determinationof the expression level of liver fibrosis related nucleic acid sequencesby the methods outlined above. The availability of antibodies andaptamers specific to liver fibrosis related protein(s) facilitates thedetection and quantification of such protein(s) by one of a number ofimmunoassay methods that are well known in the art, such as thosepresented in Harlow and Lane (Antibodies, A Laboratory Manual, CSHL, NewYork, 1988). In addition, if such antibodies are not available, methodsof constructing antibodies are routine in the art.

TABLE 7 Exemplary antibodies Protein Source Catalog Number(s) F2(Prothrombin) Thermo Fisher PA1-74072, PA1-74070, Scientific IncPA1-74127 C4A (Complement 4A) Thermo Fisher PA5-16602, LF-MA0187,Scientific Inc LF-MA0188, PA1-27058, PA1-28407 QSOX Thermo FisherPA5-21578 (Sulfhydrul oxidase 1) Scientific Inc ECM1 Thermo FisherMA5-12004, MA1-19051 Scientific Inc LGALS3BP Biorbyt orb101823,orb102911

Any standard immunoassay format (such as ELISA, Western blot, RIA assay,or lateral flow device) can be used to measure liver fibrosis relatedprotein levels. A comparison to control (e.g., subject who does not haveliver fibrosis or who has non-progressing liver fibrosis) and anincrease or decrease in liver fibrosis related protein expression levels(such as an increase or decrease in any combination of at least 4, atleast 5, at least 6, at least 8, or at least 10 proteins or peptideslisted in any of Tables 1, 2, 3, 5, 6 and 12, as well as any of SEQ IDNOS: 1-5544) is indicative of liver fibrosis. Immunohistochemicaltechniques can also be utilized for protein detection andquantification. For example, a sample can be obtained from a subject,and stained for the presence of a liver fibrosis related protein usingthe appropriate protein specific binding agents and any standarddetection system (such as one that includes a secondary antibodyconjugated to horseradish peroxidase). General guidance regarding suchtechniques can be found in Bancroft and Stevens (Theory and Practice ofHistological Techniques, Churchill Livingstone, 1982) and Ausubel et al.(Current Protocols in Molecular Biology, John Wiley & Sons, New York,1998).

For the purposes of quantifying liver fibrosis related proteins, abiological sample of the subject that includes cellular proteins can beused. Quantification of a liver fibrosis related protein can be achievedby immunoassay and the amount compared to levels of the protein found incells from a subject who does not have liver fibrosis or hasnon-progressing liver fibrosis.

In one example, a spectrometric method is utilized to detect or quantifyan expression level of a target protein (such as those in any of Tables1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544). Exemplaryspectrometric methods include mass spectrometry, nuclear magneticresonance spectrometry, and combinations thereof. In one example, massspectrometry is used to detect the presence of a target protein (such asthose in any of Tables 1-3, 5-6, 12-24) in a biological sample (see forexample, Stemmann et al., Cell 107(6):715-26, 2001; Zhukov et al., “FromIsolation to Identification: Using Surface Plasmon Resonance-MassSpectrometry in Proteomics, PharmaGenomics, March/April 2002).

A target protein (such as a liver fibrosis related protein or peptide)also can be detected by mass spectrometry assays coupled toimmunaffinity assays, the use of matrix-assisted laserdesorption/ionization time-of-flight (MALDI-TOF) mass mapping and liquidchromatography/quadrupole time-of-flight electrospray ionization tandemmass spectrometry (LC/Q-TOF-ESI-MS/MS) sequence tag of proteinsseparated by two-dimensional polyacrylamide gel electrophoresis(2D-PAGE) (Kiernan et al., Anal. Biochem., 301: 49-56, 2002; Poutanen etal., Mass Spectrom., 15: 1685-1692, 2001).

Quantitative mass spectroscopic methods, such as SELDI, can be used toanalyze protein expression in a sample. In one example, surface-enhancedlaser desorption-ionization time-of-flight (SELDI-TOF) mass spectrometryis used to detect protein expression, for example by using theProteinChip™ (Ciphergen Biosystems, Palo Alto, Calif.). An Agilent 6224TOF Mass Spectrometer, which may be upgraded to a 1.5 meter flight tubeproviding resolution of ˜25,000, can be used to detect proteinexpression. Similar methods are known in the art (for example see U.S.Pat. No. 5,719,060; U.S. Pat. No. 6,897,072; and U.S. Pat. No.6,881,586). SELDI is a solid phase method for desorption in which theanalyte is presented to the energy stream on a surface that enhancesanalyte capture or desorption. In one embodiment, mass spectrometry iscoupled to ion mobility separation (IMS-MS) and optionally, liquidchromatography, for the detection of protein expression. In anembodiment, a single instrument couples ion mobility separation (IMS-MS)to mass spectrometry for the detection of liver fibrosis related proteinexpression. In one embodiment, the instrument couples 1-m ion mobilityseparation (IMS-MS) with a time-of-flight mass spectrometer such as theAgilent 6224 TOF MS which may optionally be upgraded to a 1.5 meterflight tube. In one embodiment, IMS separations that take place on thetime scale of tens of milliseconds offer an additional separation stageand a way of reducing the need for extended LC separation times. In anIMS separation, ions subject to an electric field while travelingthrough a buffer gas separate quickly based on ion shape, e.g. compactspecies drift faster than those with extended structures [9, 10]. IMScan be coupled between LC and orthogonal acceleration time-of-flight(TOF) MS stages, and by combining the three orthogonal separations intoa single LC-IMS-MS instrumentation platform, multidimensionalhigh-resolution nested spectra are produced containing elution times,mass-to-charge ratios (m/z) and IMS drift times for all detectable ionsin a sample [11, 12]

In one example, one or more of the liver fibrosis related proteins orpeptides listed in any of Tables 1, 2, 3, 5, 6 and 12, as well as any ofSEQ ID NOS: 1-5544 are detected using a tandem mass spectrometrytechnique, for example by spiking one or more stable isotope peptides tobe detected (such as one or more of those in Tables 1, 2, 3, 5, 6 and12, as well as any of SEQ ID NOS: 1-5544) into a test serum or plasmasample and detecting setting parameters for the specific detection andaccurate quantification of the desired liver fibrosis related proteins.

Briefly, one version of SELDI uses a chromatographic surface with achemistry that selectively captures analytes of interest, such as fouror more those in any of Tables 1, 2, 3, 5, 6 and 12, as well as any ofSEQ ID NOS: 1-5544. Chromatographic surfaces can be composed ofhydrophobic, hydrophilic, ion exchange, immobilized metal, or otherchemistries. For example, the surface chemistry can include bindingfunctionalities based on oxygen-dependent, carbon-dependent,sulfur-dependent, and/or nitrogen-dependent means of covalent ornoncovalent immobilization of analytes. The activated surfaces are usedto covalently immobilize specific “bait” molecules such as antibodies,receptors, or oligonucleotides often used for biomolecular interactionstudies such as protein-protein and protein-DNA interactions.

The surface chemistry allows the bound analytes to be retained andunbound materials to be washed away. Subsequently, analytes bound to thesurface (such as any of those in Tables 1, 2, 3, 5, 6 and 12, as well asany of SEQ ID NOS: 1-5544) can be desorbed and analyzed by any ofseveral means, for example using mass spectrometry. When the analyte isionized in the process of desorption, such as in laserdesorption/ionization mass spectrometry, the detector can be an iondetector. Mass spectrometers generally include means for determining thetime-of-flight of desorbed ions. This information is converted to mass.However, one need not determine the mass of desorbed ions to resolve anddetect them: the fact that ionized analytes strike the detector atdifferent times provides detection and resolution of them.Alternatively, the analyte can be detectably labeled (for example with afluorophore or radioactive isotope). In these cases, the detector can bea fluorescence or radioactivity detector. A plurality of detection meanscan be implemented in series to fully interrogate the analyte componentsand function associated with retained molecules at each location in thearray.

Therefore, in a particular example, the chromatographic surface includesantibodies that specifically bind a target protein (such as one or moreof those in any of Tables 1, 2, 3, 5, 6 and 12, as well as any of SEQ IDNOS: 1-5544). In other examples, the chromatographic surface consistsessentially of, or consists of, antibodies that specifically bind atarget protein (such as those in any of Tables 1, 2, 3, 5, 6 and 12, aswell as any of SEQ ID NOS: 1-5544). In some examples, thechromatographic surface includes antibodies that bind other molecules,such as housekeeping proteins.

In another example, antibodies are immobilized onto the surface using abacterial Fc binding support. The chromatographic surface is incubatedwith a sample. The antigens present in the sample can recognize theantibodies on the chromatographic surface. The unbound proteins and massspectrometric interfering compounds are washed away and the proteinsthat are retained on the chromatographic surface are analyzed anddetected by SELDI-TOF. The MS profile from the sample can be thencompared using differential protein expression mapping, whereby relativeexpression levels of proteins at specific molecular weights are comparedby a variety of statistical techniques and bioinformatic softwaresystems.

Alternatively, the amount of a target protein (such as a liver fibrosisrelated protein) can be determined using fluorescent methods. In oneembodiment, the activity of a fibrosis related protein is measured usinga substrate which may emit fluorescence when cleaved by the fibrosisrelated protein. Alternatively, the activity of a fibrosis relatedprotein may be measured using a fluorescent substrate whose fluorescenceis quenched when cleaved by the fibrosis related protein.

To provide an example of a fluorescent method, Quantum dots (Qdots®) areuseful, for example when using immunohistochemistry, flow cytometry, andplate-based assays, and may therefore be used in conjunction with thisdisclosure. Qdot® nanocrystals have unique optical properties includingan extremely bright signal for sensitivity and quantitation; and highphotostability for imaging and analysis. A single excitation source isneeded, and a growing range of conjugates makes them useful in a widerange of cell-based applications. Qdot® Bioconjugates are characterizedby quantum yields comparable to the brightest traditional dyesavailable. Additionally, these quantum dot-based fluorophores absorb10-1000 times more light than traditional dyes. The emission from theunderlying Qdot® quantum dots is narrow and symmetric, which meansoverlap with other colors is minimized, resulting in minimal bleedthrough into adjacent detection channels and attenuated crosstalk, inspite of the fact that many more colors can be used simultaneously.Standard fluorescence microscopes are an inexpensive tool for detectingQdot® Bioconjugates. Since Qdot® conjugates are virtually photo-stable,time can be taken with the microscope to find regions of interest andadequately focus on the samples. Qdot® conjugates are useful any timebright photo-stable emission is required and are particularly useful inmulticolor applications where only one excitation source/filter isavailable and minimal crosstalk among the colors is required.

For example, Qdot® Fluorescent IHC can be performed with secondaryantibodies, where the detection substrates are streptavidin-conjugatedQdots®. Image analysis can be performed by initially capturing imagecubes on a spectral imaging camera (Cambridge Research Instruments,Woburn, Mass.). Excitation can be conducted with a UV (mercury) lightsource. The image cubes can then analyze. Briefly, image cubes can beretrieved in the application and data can be extracted and reportedbased on the pixel intensities of Qdots® expected to emit at 605 nm and655 nm.

As an example, fluorescence can be measured with the multispectralimaging system Nuance™ (Cambridge Research & Instrumentation, Woburn,Mass.). As another example, fluorescence can be measured with thespectral imaging system SpectrView™ (Applied Spectral Imaging, Vista,Calif.). Multispectral imaging is a technique in which spectroscopicinformation at each pixel of an image is gathered and the resulting dataanalyzed with spectral image-processing software. For example, theNuance system can take a series of images at different wavelengths thatare electronically and continuously selectable and then utilized with ananalysis program designed for handling such data. The Nuance system isable to obtain quantitative information from multiple dyessimultaneously, even when the spectra of the dyes are highly overlappingor when they are co-localized, or occurring at the same point in thesample, provided that the spectral curves are different. Many biologicalmaterials autofluorescence, or emit lower-energy light when excited byhigher-energy light. This signal can result in lower contrast images anddata. High-sensitivity cameras without multispectral imaging capabilityonly increase the autofluorescence signal along with the fluorescencesignal. Multispectral imaging can unmix, or separate out,autofluorescence from the sample and, thereby, increase the achievablesignal-to-noise ratio.

D. Solid Supports or Substrates

The described techniques for detecting liver fibrosis related molecules,including proteins and nucleic acids, may include a solid support orsubstrate which is insoluble or can be made insoluble by a subsequentreaction. Numerous and varied solid supports are known to those skilledin the art and include, without limitation, nitrocellulose, the walls ofwells of a reaction tray, multi-well plates, test tubes, polystyrenebeads, magnetic beads, membranes and microparticles (such as latexparticles). Any suitable porous material with sufficient porosity toallow access by detector reagents and a suitable surface affinity toimmobilize capture reagents (e.g., lectins or antibodies) iscontemplated by this term. For example, the porous structure ofnitrocellulose has excellent absorption and adsorption qualities for awide variety of reagents, for instance, capture reagents. Nylonpossesses similar characteristics and is also suitable. Microporousstructures are useful, as are materials with gel structure in thehydrated state.

Further examples of useful solid supports include: natural polymericcarbohydrates and their synthetically modified, cross-linked orsubstituted derivatives, such as agar, agarose, cross-linked alginicacid, substituted and cross-linked guar gums, cellulose esters,especially with nitric acid and carboxylic acids, mixed celluloseesters, and cellulose ethers; natural polymers containing nitrogen, suchas proteins and derivatives, including cross-linked or modifiedgelatins; natural hydrocarbon polymers, such as latex and rubber;synthetic polymers which may be prepared with suitably porousstructures, such as vinyl polymers, including polyethylene,polypropylene, polystyrene, polyvinylchloride, polyvinylacetate and itspartially hydrolyzed derivatives, polyacrylamides, polymethacrylates,copolymers and terpolymers of the above polycondensates, such aspolyesters, polyamides, and other polymers, such as polyurethanes orpolyepoxides; porous inorganic materials such as sulfates or carbonatesof alkaline earth metals and magnesium, including barium sulfate,calcium sulfate, calcium carbonate, silicates of alkali and alkalineearth metals, aluminum and magnesium; and aluminum or silicon oxides orhydrates, such as clays, alumina, talc, kaolin, zeolite, silica gel, orglass (these materials may be used as filters with the above polymericmaterials); and mixtures or copolymers of the above classes, such asgraft copolymers obtained by initializing polymerization of syntheticpolymers on a pre-existing natural polymer.

It is contemplated that porous solid supports, such as nitrocellulose,described herein can be in the form of sheets or strips. The thicknessof such sheets or strips may vary within wide limits, for example, fromabout 0.01 to 0.5 mm, from about 0.02 to 0.45 mm, from about 0.05 to 0.3mm, from about 0.075 to 0.25 mm, from about 0.1 to 0.2 mm, or from about0.11 to 0.15 mm. The pore size of such sheets or strips may similarlyvary within wide limits, for example from about 0.025 to 15 microns, ormore specifically from about 0.1 to 3 microns; however, pore size is notintended to be a limiting factor in selection of the solid support.

The surface of a solid support may be activated by chemical processesthat cause covalent linkage of an agent (e.g., a capture reagent, suchas an antibody) to the support. However, any other suitable method maybe used for immobilizing an agent (e.g., a capture reagent) to a solidsupport including, without limitation, ionic interactions, hydrophobicinteractions, covalent interactions and the like. The particular forcesthat result in immobilization of an agent on a solid phase are notimportant for the methods and devices described herein.

A solid phase can be chosen for its intrinsic ability to attract andimmobilize an agent, such as a capture reagent (such as an antibody oroligonucleotide probe). Alternatively, the solid phase can possess afactor that has the ability to attract and immobilize an agent, such asa capture reagent. The factor can include a charged substance that isoppositely charged with respect to, for example, the capture reagentitself or to a charged substance conjugated to the capture reagent. Inanother embodiment, a specific binding member may be immobilized uponthe solid phase to immobilize its binding partner (e.g., a capturereagent). In this example, therefore, the specific binding memberenables the indirect binding of the capture reagent to a solid phasematerial.

Except as otherwise physically constrained, a solid support may be usedin any suitable shapes, such as films, sheets, strips, or plates, or itmay be coated onto or bonded or laminated to appropriate inert carriers,such as paper, glass, plastic films, or fabrics.

E. Lateral Flow Devices

The described techniques for detecting liver fibrosis related molecules,including proteins and nucleic acids, may include use of a lateral flowdevice. A lateral flow device may be an analytical device in the form ofa test strip used in lateral flow chromatography, in which a samplefluid, such as one to be tested for the presence of liver fibrosisrelated molecules (such as four or more of those shown in any of Tables1, 2, 3, 5, 6 and 12, as well as any of SEQ ID NOS: 1-5544), flows (forexample by capillary action) through the strip (which is frequently madeof bibulous materials such as paper, nitrocellulose, and cellulose). Thetest sample and any suspended target molecule(s) can flow along thestrip to a detection zone to indicate a presence, absence and/orquantity of the target molecule(s)

Numerous lateral flow analytical devices are known, and include thoseshown in U.S. Pat. Nos. 4,313,734; 4,435,504; 4,775,636; 4,703,017;4,740,468; 4,806,311; 4,806,312; 4,861,711; 4,855,240; 4,857,453;4,943,522; 4,945,042; 4,496,654; 5,001,049; 5,075,078; 5,126,241;5,451,504; 5,424,193; 5,712,172; 6,555,390; 6,368,876; 7,799,554; EP0810436; and WO 92/12428; WO 94/01775; WO 95/16207; and WO 97/06439.

Lateral flow devices can in one example be a one-step lateral flow assayin which a sample fluid is placed in a sample or wicking area on abibulous strip (though, non-bibulous materials can be used, and renderedbibulous by applying a surfactant to the material), and allowed tomigrate along the strip until the sample comes into contact with thedetection mechanism. In one embodiment, the sample fluid can flow intoan area of the lateral flow device housing oligonucleotide probes forthe at least two (e.g., at least 3, at least 4, at least 5 or at least10) liver fibrosis related nucleic acids. In another embodiment, thesample fluid can flow into an area of the lateral flow device housingantibodies specific for the at least two (e.g., at least 3, at least 4,at least 5 or at least 10) liver fibrosis related proteins and,optionally, labeled secondary antibodies specific for the antibodieswhich bind to the at least two (e.g., at least 3, at least 4, at least 5or at least 10) liver fibrosis related proteins.

In some examples, the strip includes multiple regions for detectingdifferent target proteins or other molecules in the sample (for examplein parallel lines or as other separate portions of the device). Forexample, the device can include different regions, each containingantibodies or oligonucleotide probes specific for a different liverfibrosis-related proteins or nucleic acids, respectively. For example,the device can include at least 4, at least 10, at least 20, at least50, or at least 100 different such regions. The test strips can alsoincorporate control indicators, which provide a signal that the test hasadequately been performed, even if a positive signal indicating thepresence (or absence) of a target is not achieved.

A lateral flow device can include a sample application area or wickingpad, which is where the fluid or liquid sample is introduced. In oneexample, the sample may be introduced to the sample application area byexternal application, as with a dropper or other applicator. In anotherexample, the sample application area may be directly immersed in thesample, such as when a test strip is dipped into a container holding asample. In yet another example, the sample may be applied, blotted,poured or expressed onto the sample application area.

A lateral flow device can include a reagent or conjugation pad, theregion of a lateral flow device where reagents are immobilized, such asantibodies or aptamers for liver fibrosis-related proteins, which may beimmobilized to magnetic beads or other materials. A lateral flow devicemay have more than one conjugation area, for example, a “primaryconjugation area,” a “secondary conjugation area,” and so on. Oftendifferent reagents are immobilized in the primary, secondary, or otherconjugation areas. Multiple conjugation areas may have any orientationwith respect to each other on the lateral flow substrate; for example, aprimary conjugation area may be distal or proximal to a secondary (orother) conjugation area and vice versa. Alternatively, a primaryconjugation area and a conjugation (or other) area may be orientedperpendicularly to each other such that the two (or more) conjugationareas form a cross or a plus sign or other symbol. For example, Apiluxet al. (Anal. Chem. 82:1727-32, 2010), Dungchai et al. (Anal. Chem.81:5821-6, 2009), and Dungchai et al. (Analytica Chemica Acta674:227-33, 2010), provide exemplary lateral flow devices with a centralsample area and one or more conjugation areas distal to the sample area,which provide independent test zones where independent reactions canoccur (e.g., each test zone has a different reagents for detecting aparticular liver fibrosis-related molecule, and can further include oneor more reaction pads where reactions can take place (for exampleinterspersed between the reagent pads) and an absorption pad, forexample that form a “Y”, cloverleaf, or spoke-wheel pattern.

A lateral flow device can include one or more reaction pads, such as amembrane, that can be placed to allow desired reactions to occur, and anabsorption pad that draws the sample across the conjugation pad(s) andmembrane(s) by capillary action and collects it.

F. ELISA Plates

In the most common type of ELISA, the solid phase is coated with amember of the binding pair. Thus, ELISA plates containing a specificbinding agent for each of the at least two liver fibrosis relatedmolecules (e.g., at least 3, at least 4, at least 5 or at least 10) tobe detected, each in different wells or different sections of the plate,can be used. For example, the solid phase can be coated with antibodiesor aptamers specific for the liver fibrosis-related proteins to bedetected, each on a different region of the solid phase. For example,ELISA plates can be coated with a specific antibody (or antigen). Themicroplate may be incubated for a period of time, during which time theantibodies or aptamers adhere to the walls of the microwells up to thefluid fill level. The microwells are then washed leaving a microplatehaving microwells whose walls are uniformly covered with antibodies oraptamers up to the fluid fill level. Similarly, the solid phase can becoated with oligonucleotides specific for the liver fibrosis-relatednucleic acids to be detected, each on a different region of the solidphase.

In some examples, an aliquot of the sample to be examined is incubatedwith the solid coated solid phase and any liver fibrosis-relatedmolecule that may be present is captured onto the solid phase. Afterwashing to remove residual sample and any interfering materials it maycontain, a second binding agent, specific for the liver fibrosis-relatedmolecule and conjugated to label can be added to the solid phase topermit detection of any liver fibrosis-related molecule that bound tothe agents immobilized on the solid phase. Alternatively, the liverfibrosis-related molecules in the sample are labeled prior to applyingthem to the solid phase, thus permitting their detection upon binding.

It will be realized that the above is a general procedure for bioassayand that many variations are known in the art including fluorogenic andluminogenic substrates for ELISA, direct labeling of the second memberof the binding pair with a fluorescent or luminescent molecule andnucleic acids or other specific pairing agents instead of antibodies asthe binding agent. Samples may be diluted prior to being dispensed intothe solid phase or they may be dispensed into deep well microplates,diluted in situ and then the diluted analyte transferred to thefunctional solid phase.

The most common type of solid phase is a standard sample vessel known asa microplate which can be stored easily and which may be used with avariety of biological specimens. Microplates are available commerciallyand are made from e.g., polystyrene, PVC, Perspex or Lucite Knownmicroplates comprise 96 wells (also commonly known as “microwells”)which are symmetrically arranged in an 8×12 array. Microwells typicallyhave a maximum volume capacity of approximately 350 μl. However,normally only 10-200 μl of fluid is dispensed into a microwell. In somearrangements of the microplate the microwells are arranged in strips of8 or 12 wells that can be moved and combined in a carrier to give acomplete plate having conventional dimensions. One skilled in the artwill appreciate that other arrangements of microplates are known.

Positive and negative controls are generally supplied with commercialkits and are used for quality control and to provide a relative cut-off.After reading the processed microplate, the results of the controls arechecked against the manufacturer's validated values to ensure that theanalysis has operated correctly and then the value is used todistinguish positive from negative specimens and a cut-off value iscalculated. Standards are usually provided for quantitative assays andare used to build a standard curve from which the concentration ofanalyte in a specimen may be interpolated.

It will be recognized that the ELISA procedure as outlined aboveinvolves multiple steps including pipetting, incubation, washing,transferring microplates between activities, reading and data analysis.These steps can be automated.

A laboratory can receive a number of sample tubes containing, forexample, body fluid from a number of patients. A specified amount offluid is then removed from the sample and is then dispensed into one ormore microwells of a microplate containing the desired specific bindingagents (e.g., antibodies, aptamers, oligonucleotide probes). To detectseveral different proteins, the sample is dispensed into a number ofseparate microplates or microwells, each coated with a different bindingagent. Alternatively, a single microplate can contain a plurality ofwells, each coated with the same specific binding agent (or having a fewwells as controls), thus permitting analysis of a plurality of differentpatient samples. Each microplate or microwell can then be processed todetect the presence of a different liver fibrosis-related molecule. Toanalyze several different liver fibrosis-related molecules amultiplicity of microplates can be used and transfer of aliquots of thesame specimen to the different microplates. Similarly, use ofmicroplates with different binding agents in different microwellspermits mutiplexing. For example, a single microwell may be used todetermine whether a patient has antibodies to one of the at least two(e.g., at least 3, at least 4, at least 5 or at least 10) liver fibrosisrelated proteins.

Multiplexing enables multiple different tests to be performedsimultaneously upon the same patient sample. One approach tomultiplexing is to provide a microplate comprising 96 sample wellswherein an array of different capture antibodies is disposed in eachsample well. The arrays comprise, for example, 20 nl spots each having adiameter of 350 μm. The spots are arranged with a pitch spacing of, forexample, 650 μm. Each spot may correspond with a different captureantibody. Multiplexing enables a greater number of data points and moreinformation per assay to be obtained compared with conventional ELISAtechniques wherein each sample plate tests for a single analyte ofinterest.

VI. Treatment of Liver Fibrosis and Related Conditions

In certain embodiments, a therapy may be provided based on the diagnosisor prognosis of liver fibrosis or pre-fibrosis. Thus, in some examplesthe disclosed methods include treating a subject who has been diagnosedor prognosed with liver fibrosis. The disclosed therapeutic agents areadministered at therapeutically effective amounts.

Treatment, as used herein, refers to any therapeutic intervention thatameliorates a sign or symptom of a pathological condition, such a signor symptom of liver fibrosis, or interferes with an underlyingpathological process implicated in the condition, such as inflammationin the liver, elevation of liver enzyme levels, and/or the formation offibrotic structures within the liver. Treatment can also induceremission or cure of a condition, such as liver fibrosis. In particularexamples, treatment includes preventing a disease, for example byinhibiting the full development of a disease, such as preventing liverfibrosis or preventing development of a disease or disorder that resultsfrom a liver fibrosis. Prevention of a disease does not require a totalabsence of disease. For example, a decrease of at least 20% or at least50% can be sufficient.

Treatment of liver fibrosis may be directed toward treatment of liverfibrosis itself and/or treating an underlying cause of liver fibrosis.Recent evidence indicates that even advanced fibrosis is reversible. Inexperimentally induced fibrosis, cessation of liver injury results infibrosis regression. In humans, spontaneous resolution of liver fibrosiscan occur after successful treatment of the underlying disease. In oneexample, the antifibrotic therapy administered to the subject isliver-specific, well tolerated when administered for prolonged periodsof time, and effective in attenuating excessive collagen depositionwithout affecting normal ECM synthesis. Exemplary therapies that can beadministered in therapeutically effective amounts to a subject diagnosedor prognosed with liver fibrosis are shown in Table 8.

TABLE 8 Exemplary anti-fibrotic drugs for the treatment of liverfibrosis Agent Mode of Administration Angiotensin inhibitors iv, oralColchicine Iv, oral Corticosteroids iv, oral Endothelin inhibitors iv,oral Interferon-alpha iv, oral Interleukin-10 iv Pentoxyfylline iv, oralPhosphatidylcholine iv, oral PPAR antagonists iv, oral S-adenosylmethionine iv, oral Sho-saiko-to oral TGF-β1 inhibitors iv, oralTocopherol iv, oral

In one example, an antiinflammatory drug is used to treat or prevent theliver fibrosis. Corticosteroids can be administered to treat hepaticfibrosis in patients with autoimmune hepatitis and acute alcoholichepatitis.

Inhibition of the accumulation of activated hepatic stellate cells(HSCs) by modulating either their activation and/or proliferation orpromoting their apoptosis is another treatment method. Antioxidants suchas vitamin E, silymarin, phosphatidylcholine, andS-adenosyl-L-methionine inhibit HSC activation, protect hepatocytes fromundergoing apoptosis, and attenuate experimental liver fibrosis.Antioxidants exert beneficial effects in patients with liver fibrosispatients such as those with alcohol-induced liver disease and NASH.Cell-specific delivery to HSCs can be obtained using different carriers(e.g., cyclic peptides coupled to albumin recognizing collagen type VIreceptor and/or PDGFR).

In one example, treatment or prevention of liver fibrosis includesadministration of an agent that disrupts TGF-β synthesis and/orsignaling pathways. In one example, treatment of prevention of liverfibrosis includes administration of a growth factor (e.g., IGF,hepatocyte growth factor, and cardiotrophin) to attenuate liverfibrosis. Substances that inhibit key signal transduction pathwaysinvolved in liver fibrogenesis, such aspentoxifylline (phosphodiesteraseinhibitor), amiloride (Na⁺/H⁺ pump inhibitor), andS-farnesylthiosalicylic acid (Ras antagonist), can be used. In addition,ligands of PPARa and/or PPARy such as thiazolindiones can be used totreat liver fibrosis.

In one example, inhibition of the renin-angiotensin system is used totreat liver fibrosis.

The blockade of endothelin-1 type A receptors and the administration ofvasodilators (prostaglandin E2 and nitric oxide donors) exertantifibrotic activity. Different herbal compounds, such as Sho-saiko-to,glycyrrhizin, and savia miltiorhiza, have antifibrotic effects. Anotherapproach is the inhibition of collagen production and/or the promotionof its degradation. For example, inhibitors of prolyl-4 hydroxylase andhalofuginone can prevent the development of cirrhosis by inhibitingcollagen synthesis. MMP-8 and urokinase-type plasminogen activator canalso stimulate collagen degradation. In one example, infusion ofmesenchymal stem cells ameliorates fibrosis.

Antifibrotic therapy may differ depending on the type of liver disease.In patients with chronic HCV infection, antiviral treatments (e.g.,pegylated IFN plus ribavirin) can improve liver fibrosis.Renin-angiotensin system inhibitors can be used for the treatment ofliver fibrosis in patients with chronic HCV infection. In some examples,treatment of metabolic syndrome in patients with chronic hepatitis C mayalso decrease fibrosis progression. In patients with alcohol-inducedliver disease, alcohol abstinence may be prescribed forprevention/treatment of liver fibrosis. Antioxidants (e.g.,S-adenosyl-L-methionine and phosphatidylcholine) and hepatocyteprotectors (e.g., silymarin) slow down the progression of liver fibrosisand can improve survival. In some examples, for example in patients withautoimmune hepatitis, immunosuppressant therapy exerts antifibroticeffects. Ursodeoxycholic acid can be administered to treat or preventliver fibrosis. In patients with NASH, weight loss and specifictreatments of the metabolic syndrome can reduce fibrosis development.Antioxidants and insulin sensitizers (e.g., thiazolindiones) can be usedto exert antifibrogenic effects. Use of pluripotential stem cells inhepatic wound healing can be an effective treatment for liver fibrosis.

Autoimmune hepatitis may be treated with immunosuppressiveglucocorticoids with or without azathioprine and thus prevent liverfibrosis development in these patients. Budesonide can induce remissionof autoimmune hepatitis with fewer adverse effects than, for example,prednisone. Subjects who do not respond to glucocorticoids andazathioprine may be given other immunosuppressives such asmycophenolate, cyclosporin, tacrolimus and methotrexate.

Kits

The present disclosure provides for kits that include reagents that canbe used to diagnose or prognose liver fibrosis, for example to determineif a subject has liver fibrosis or has an increased predisposition todeveloping liver fibrosis. Such kits allow one to determine if a subjecthas a differential expression in four or more liver fibrosis relatedmolecules, such as any of those listed in Tables 1, 2, 3, 5, 6 and 12,as well as any of SEQ ID NOS: 1-5544. In some examples, the kit includesa lateral flow device or ELSIA plate containing binding agents specificfor at least two (e.g., at least 3, at least 4, at least 5 or at least10) different liver fibrosis-related molecules, such as antibodies,aptamers, or oligonucleotide probes.

In one example the disclosed kits include binding molecules such asoligonucleotide probes, oligonucleotide primers, antibodies, aptamers oraptazymes (or combinations thereof) that selectively hybridize or bindto liver fibrosis-related molecules that are the target of the kit. Sucholigonucleotide probes, oligonucleotide primers, antibodies, aptamers oraptazymes can include a detectable label, such as a fluorophore. Inaddition, the oligonucleotide probes, oligonucleotide primers,antibodies, aptamers or aptazymes can be in separate vials orcontainers. In some examples, the oligonucleotide probes,oligonucleotide primers, antibodies, aptamers or aptazymes are part of asolid substrate, such as a lateral flow device or ELISA plate.

In one embodiment, the disclosed kits include oligonucleotide probes orprimers (such as a pair of primers that permits amplification of atarget liver fibrosis related nucleic acid) that can hybridize to atleast two (e.g., at least 3, at least 4, at least 5 or at least 10)different liver fibrosis related nucleic acid molecules (such as mRNA orcDNA) under high stringency. In some examples such probes or primers areat least 10 nucleotides in length, such as at least 12, at least 15, atleast 20, at least 30, or at least 50 nucleotides in length. In someexamples, the kit includes both oligonucleotide primers for amplifyingat least two (e.g., at least 3, at least 4, at least 5 or at least 10)different liver fibrosis related nucleic acid molecules, andcorresponding oligonucleotide probes to detect the resulting amplicons.The disclosed kits may further include a substrate to which theoligonucleotide probes are attached.

In one embodiment, the disclosed kits include antibodies (or aptamers oraptazymes) that can specifically bind to at least two (e.g., at least 3,at least 4, at least 5 or at least 10) different liver fibrosis-relatedproteins. Such kits may additionally include labeled secondaryantibodies specific for the primary antibodies which bind to one or moreof the at least two (e.g., at least 3, at least 4, at least 5 or atleast 10) liver fibrosis related proteins. The disclosed kits mayfurther include a substrate to which the antibodies are attached.

In particular examples, the oligonucleotide probes or antibodies areattached to an array, such as a biochip, lateral flow device, ordipstick. Such an array can include other oligonucleotides orantibodies, for example to serve as negative or positive controls. Inone example, the kit includes oligonucleotide probes or primers (orantibodies) that recognize any combination of at least two of theproteins or peptides in any of Tables 1, 2, 3, 5, 6 and 12, as well asany of SEQ ID NOS: 1-5544, such as at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, at least 10, at least11, at least 12, at least 13, at least 14, at least 15, at least 16, atleast 17, at least 18, at least 19, at least 20, at least 21, at least22, at least 23, at least 24, at least 25, at least 26, at least 27, atleast 28, at least 29, at least 31, at least 31, at least 32, at least33, at least 34, at least 35, at least 36, at least 37, at least 38, atleast 39, at least 40, at least 41, at least 42, at least 43, at least44, at least 45, at least 46, at least 47, at least 48, at least 49, atleast 50, at least 51, at least 52, at least 53, at least 54, at least55, at least 56, at least 57, at least 58, at least 59, at least 60, atleast 61, at least 62, at least 63, at least 64, at least 65, at least66, at least 67, at least 68, at least 69, at least 70, at least 71, atleast 72, at least 73, at least 74, at least 75, at least 76, at least77, at least 78, at least 79, at least 80, at least 81, at least 82, atleast 83, at least 84, at least 85, at least 86, at least 87, at least88, at least 89, at least 90, at least 91, at least 92, at least 93, atleast 94, at least 95, at least 96, at least 97, at least 98, at least99, at least 100, at least 101, at least 102, at least 103, at least104, at least 105, at least 106, at least 107, at least 108, at least109, at least 110, at least 111, at least 112, at least 113, at least114, at least 115, at least 116, at least 117, at least 118, at least119, at least 120, at least 121, at least 122, at least 123, at least124, at least 125, at least 126, at least 127, at least 128, at least129, at least 130, at least 131, at least 132, at least 133, at least134, at least 135, or all 136 of the liver fibrosis related proteinslisted in Tables 1. In various examples, the kit includesoligonucleotide probes or primers (or antibodies) that recognize atleast two (e.g., at least 3, at least 4, at least 5 or at least 10)proteins or peptides listed in one of Table 2, Table 3, Table 5, or anyof SEQ ID NOS: 1-5544.

In another example, the kit includes one or more of:

1) at least two stable isotope labeled liver fibrosis proteins listed inTable 1, such as at least 3, at least 4, at least 5, at least 6, atleast 7, at least 8, at least 9, at least 10, at least 11, at least 12,at least 13, at least 14, at least 15, at least 16, at least 17, atleast 18, at least 19, at least 20, at least 21, at least 22, at least23, at least 24, at least 25, at least 26, at least 27, at least 28, atleast 29, at least 31, at least 31, at least 32, at least 33, at least34, at least 35, at least 36, at least 37, at least 38, at least 39, atleast 40, at least 41, at least 42, at least 43, at least 44, at least45, at least 46, at least 47, at least 48, at least 49, at least 50, atleast 51, at least 52, at least 53, at least 54, at least 55, at least56, at least 57, at least 58, at least 59, at least 60, at least 61, atleast 62, at least 63, at least 64, at least 65, at least 66, at least67, at least 68, at least 69, at least 70, at least 71, at least 72, atleast 73, at least 74, at least 75, at least 76, at least 77, at least78, at least 79, at least 80, at least 81, at least 82, at least 83, atleast 84, at least 85, at least 86, at least 87, at least 88, at least89, at least 90, at least 91, at least 92, at least 93, at least 94, atleast 95, at least 96, at least 97, at least 98, at least 99, at least100, at least 101, at least 102, at least 103, at least 104, at least105, at least 106, at least 107, at least 108, at least 109, at least110, at least 111, at least 112, at least 113, at least 114, at least115, at least 116, at least 117, at least 118, at least 119, at least120, at least 121, at least 122, at least 123, at least 124, at least125, at least 126, at least 127, at least 128, at least 129, at least130, at least 131, at least 132, at least 133, at least 134, at least135, or all 136 of the stable isotope labeled liver fibrosis relatedproteins listed in Table 1. In one example, the kit includes at leasttwo (e.g., at least 3, at least 4, at least 5 or at least 10) stableisotope labeled liver fibrosis proteins listed in one of Tables 2, 3,and 5, as well as any of SEQ ID NOS: 1-5544;

2) a suitable HPLC column; and

3) materials for quantifying these peptides using, for example, IMS-MS(see Examples).

Such a kit can be used to detect the at least two (e.g., at least 3, atleast 4, at least 5 or at least 10) liver fibrosis related peptides, forexample by spiking stable isotope peptides into a test serum or plasmasample and detecting setting parameters for their specific detection andaccurate quantification by, for example, using IMS-MS, MRM massspectrometry or tandem mass spectrometry. The kit can further includeone or more of a buffer solution, a conjugating solution for developingthe signal of interest, or a detection reagent for detecting the signalof interest, each in separate packaging, such as a container. Kits caninclude instructions, for instance instructions that provide calibrationcurves or charts to compare with the determined (such as experimentallymeasured) values. For example, instructions can permit the tester todetermine whether liver fibrosis related protein/peptide expressionlevels are elevated, reduced, or unchanged in comparison to a controlsample. In some examples kits include materials for obtaining a sample,such as vials, cotton swabs, and needles.

In one example, the kits can include a lateral flow device containingantibodies specific for at least two (e.g., at least 3, at least 4, atleast 5 or at least 10) different liver fibrosis related-peptides and acarrier means, such as a box, a bag, a satchel, plastic carton (such asmolded plastic or other clear packaging), wrapper (such as, a sealed orsealable plastic, paper, or metallic wrapper), or other container. Insome examples, kit components will be enclosed in a single packagingunit, such as a box or other container, which packaging unit may havecompartments into which one or more components of the kit can be placed.In other examples, a kit includes one or more containers, for instancevials, tubes, and the like that can retain, for example, one or morebiological samples to be tested, positive and/or negative controlsamples or solutions (such as, a positive control sample containing thetarget agent), diluents (such as, phosphate buffers, or saline buffers),and/or wash solutions (such as Tris buffers, saline buffer, or distilledwater).

Such kits can include other components, such as a buffer, a chart forcorrelating detected liver fibrosis related protein or nucleic acidlevel and amount of liver fibrosis related molecule present, orcombinations thereof.

Other kit embodiments include syringes, finger-prick devices, alcoholswabs, gauze squares, cotton balls, bandages, latex gloves, incubationtrays with variable numbers of troughs, adhesive plate sealers, datareporting sheets, which may be useful for handling, collecting and/orprocessing a biological sample. Kits may also optionally containimplements useful for introducing samples onto a lateral flow device,including, for example, droppers, Dispo-pipettes, capillary tubes,rubber bulbs (e.g., for capillary tubes), and the like. Still other kitembodiments may include disposal means for discarding a used deviceand/or other items used with the device (such as patient samples, etc.).Such disposal means can include, without limitation, containers that arecapable of containing leakage from discarded materials, such as plastic,metal or other impermeable bags, boxes or containers.

In some examples, a kit will include instructions for the use ofmaterials contained therein. The instructions may provide direction onhow to apply sample to the components of the kit, the amount of timenecessary or advisable to wait for results to develop, and details onhow to read and interpret the results of the test. Such instructions mayalso include standards, such as standard tables, graphs, or pictures forcomparison of the results of a test. These standards may optionallyinclude the information necessary to quantify liver fibrosis relatedmolecules.

A kit may further comprise an alarm which indicates the presence ofliver fibrosis, wherein the alarm is activated if the at least two(e.g., at least 3, at least 4, at least 5 or at least 10) proteins aredetected with elevated amounts relative to control levels such ascontrol levels for a subject without liver fibrosis. In variousembodiments, the alarm is activated if at least 70%, 75%, 80%, 85%, 90%,95% or 100% of the at least two (e.g., at least 3, at least 4, at least5 or at least 10) proteins have levels of expression that are elevatedrelative to control levels such as control levels for a subject withoutliver fibrosis. A kit may further comprise an interface which may beconfigured to accept input of a criterion for diagnosis or prognosis ofliver fibrosis. For example, a user may select the proteins for whichdifferential expression is detected and/or used to determine whether asubject has liver fibrosis. A user can also use the interface to providecriterion for a clinical judgment such as to select what percentage ofthe at least two (e.g., at least 3, at least 4, at least 5 or at least10) proteins must have differential expression to allow for a diagnosisor prognosis of liver fibrosis. In one embodiment, the alarm is a visualalarm which may be a written or digital display such as a number, word,symbolic indicator (e.g., +/−, Y, N), or combinations thereof. In anembodiment, the visual or other alarm is integrated into the interface.

EXAMPLES Example 1 Methods and Materials

This example provides technical details and procedures, includingrelevant instrument settings and materials, used to obtain the proteinexpression data from transplant and non-transplant patients discussed inthe Examples below.

Identification of Liver Fibrosis-Related Proteins in Liver TransplantSubjects

Liver transplant subjects were categorized as slow-, fast- andnon-progressors depending on the rate of fibrosis in the new liver. Eachslow and fast progressor had a matched non-progressor subject. Specimensamples were matched for the most important clinical variables known toinfluence the risk of fibrosis progression associated with recurrenthepatitis C after liver transplantation: donor age and cold ischemiatime. These pairs were also matched based on patient sex and age.Matching was also performed to time to biopsy so that there were notdiscordant times to assessment of the degree of fibrosis, as well asdays post-transplant. Patients were excluded from consideration with anyknown confounders including biliary problems, recurrent CMV infection,and more than one episode of rejection. Although there were minorvariations in the doses of immunosuppression each patient received, eachpatient was part of the same immunosuppression protocol, which includedinduction therapy, followed by tacrolimus and mycophenelate, withgradual transition to tacrolimus monotherapy after 12 months. No patientreceived more than one bolus of corticosteroids for treatment ofrejection and no patient received monoclonal antibodies (such as OKT3 orATG) for treatment of rejection.

Comparisons were performed across all slow progressors and fastprogressors compared to all their matched controls.

Identification and quantification of the detected peptide peaks wasperformed utilizing the Accurate Mass and Time (AMT) Tag approach(Zimmer et al., Mass Spectrometry Reviews, 2006. 25(3):450-482).Briefly, multiple in-house developed/publicly available informaticstools were used to process LC-MS data and correlate the resulting LC-MSfeatures to an AMT tag database containing accurate mass and LCseparation elution time information for peptide tags generated fromtandem MS Thermo LTQ Orbitrap Velos analyses of human plasma proteins.The AMT tag database only contained peptides which passed a MS-GF cutoffof 1×10⁻⁹ (Sangtae, et al., J. Proteome Res., 7 (8), 3354-3363, 2008).Among the tools used were algorithms for peak-picking and fordetermining isotopic distributions and charge states (Jaitly et al., BMCBioinformatics, 2009. 10(1):87). Further downstream data analysisincorporated all of the possible detected peptides into a visualizationprogram VIPER (Monroe, Bioinformatics, 2007. 23(15):2021-3) to correlateLC-MS features to the peptide identifications in the AMT tag database.The Viper results were matched and refined to give a median masstolerance of ±2.05 ppm and a median normalized elution tolerance (NET)of ±0.75%. VIPER provided an intensity report for all detected features,normalized LC elution times via alignment to the database, and featureidentification. A representative protein was chosen for redundantpeptides (see SEQ ID NOS: 1-2633). If a protein was identified with onlyone significant non-unique peptide it was removed. In DAnTE software,peptide peak intensity values were converted to a log 2 scalestatistically compared utilizing ANOVA (performed as a t-test with onlytwo data types in each comparisons) (Polpitiya et al., Bioinformatics,2008. 24(13):1556-8). The analysis only focused on significantlychanging peptides (p-values and q-values <0.05). The q-value of a testmeasures the proportion of false positives incurred (called the falsediscovery rate) when that particular test is called significant.Significantly changing peptides were assessed at a protein level usingDAnTE's Rollup parameters (reference peptide based scaling, wherepeptides were excluded from scaling if they were not seen in at least 3datasets and no minimum peptide presence was required). Statisticallycomparisons were also done at the protein level. Only significantlychanging proteins were retained, i.e. some peptides were significant butthey were in opposing directions, so the proteins were not significantwhen the peptides were rolled up (these opposing peptides have greycolored cells in the peptide tables). The three instrument analyses foreach same sample were averaged together to create the heatmaps depictedin FIGS. 4A and 4B. FIG. 4A contains all proteins that had at least 21datapoints and ≧2 peptides. All 136 significantly changing proteinsidentified by at least two peptides are listed in Table 1 where 34proteins increased in the slow progressors (SP) and fast progressors(FP) compared to their matched controls, 37 proteins decreased in the SPand FP compared to their matched controls, 5 proteins only increased inthe SP compared to their matched controls, 30 proteins only increased inthe FP, 19 proteins only decreased in the SP, 5 proteins only decreasedin the FP and 6 proteins increased in the FP and decreased in the SP.

A summary is provided in Table 4 above.

Identification of Liver Fibrosis-Related Proteins in Non-TransplantSubjects

In this study liver transplant patients were categorized by fibrosislevel.

Identification and quantification of the detected peptide peaks wasperformed utilizing the Accurate Mass and Time (AMT) Tag approach(Zimmer et al., Mass Spectrometry Reviews, 2006. 25(3):450-482).Briefly, multiple in-house developed/publicly available informaticstools were used to process LC-MS data and correlate the resulting LC-MSfeatures to an AMT tag database containing accurate mass and LCseparation elution time information for peptide tags generated fromtandem MS Thermo LTQ Orbitrap Velos analyses of human plasma proteins.The AMT tag database only contained peptides which passed a MS-GF cutoffof 1×10⁻⁹ (Sangtae, et al., J. Proteome Res., 7 (8), 3354-3363, 2008).Among the tools used were algorithms for peak-picking and fordetermining isotopic distributions and charge states (Jaitly et al., BMCBioinformatics, 2009. 10(1):87). Further downstream data analysisincorporated all of the possible detected peptides into a visualizationprogram VIPER (Monroe, Bioinformatics, 2007. 23(15):2021-3) to correlateLC-MS features to the peptide identifications in the AMT tag database.

Detected peptides are shown in SEQ ID NOS: 2634-5544. Significantproteins had to have ≧2 unique peptides to be used for rollup, 78proteins passed the criteria (these values were used to create theheatmap in FIG. 5). Table 12 contains the 78 significantly changingproteins and denotes if they were also observed to be differentiallyexpressed in the transplant model data.

Sample Preparation for Liver Fibrosis Samples

Human Serum Samples. Initial blood serum samples were chosen from 60HCV+ patients following liver transplantation. Additional 60non-transplant blood serum samples were obtained from HCV+ patientsthrough the Alaska Native Tribal Health Consortium (ANTHC).Non-transplant ANTHC samples were selected based upon biopsy phase(non-fibrosis, 0-1 Ishak score, versus extensive fibrosis/bridging, 4-6Ishak score), and availability and condition of serum specimen(non-thawed specimen within 6 months of a diagnostic biopsy). Allsubjects were recruited and samples were collected under institutionalreview board-approved protocols.

Serum Depletion and Protein Digestion. Individual human serum sampleswere partitioned and depleted of 14 highly abundant proteins using aProteomeLab™ 12.7×79.0-mm IgY14 LC10 affinity LC column (BeckmanCoulter, Fullerton, Calif.). The unbound, flow-through fractioncontaining low- and medium-abundance proteins was collected, directlyconcentrated in urea buffer (8M urea, 10 mM Tris-HCI, pH 7.4, 150 mMNaCI) by filter centrifugation, then denatured for 1 hour at 37° C. Allprocessing steps following depletion were carried out in a 96-well plateto minimize batch effects associated with processing, utilizingautomated protocols on a Biomek FX liquid handling robot (BeckmanCoulter). 100 μL aliquots of depleted, concentrated plasma were takenfor digestion, giving a starting amount of approximately 100 μg totalprotein. Aliquots were transferred to a 96-well plate preloaded withurea to give a concentration of 8M. Dithiothreitol (DTT) was then addedto a final concentration of 10 μM. Samples were incubated for 1 h at 37°C. to denature and reduce. Samples were then diluted 5-fold with 50 mMammonium bicarbonate prior to the addition of sequence grade trypsin(Promega) in a 1:50 enzyme to protein ratio. Enzymatic digestion wascarried out for 6 h at 37° C. Peptides were desalted using an automatedprotocol utilizing C₁₈ SPEC tips (Varian). Peptides were eluted in 200μl 80% ACN/0.1% TFA and lyophilized. Desalted peptides were rehydratedin 25 mM Ammonium bicarbonate and concentrations were determined by BCAassay. Final peptide concentrations were normalized to a finalconcentration of 0.3 μg/μL prior to storage at −80° C. until LC-MSanalysis.

Sample Preparation for Spiked Serum Sample

Eight non-plasma peptides (Table 9) from Sigma-Aldrich were used withoutfurther purification. A final concentration of 0.25 mg/mL was desiredfor the tryptically-digested human serum sample to avoid undesirableclogging effects. The serum sample was diluted to 1 mg/mL with water andthe 8 non-plasma peptides were added to specific final concentrationsfrom 100 pg/mL to 100 ng/mL (noted in Table 9). An additional standardsample consisting of the eight peptides spiked into water at the sameconcentrations as in the serum sample was prepared to determine theelution times of the spiked peptides and to compare the dynamic range ofdetection with the serum sample.

TABLE 9 Non-serum peptides spiked into human serum Spiking Level Peptide100 pg/mL Melittin, Dynorphin A Porcine Fragment 1-13  1 ng/mL Des ProAla Bradykinin, Leucine Enkephalin  10 ng/mL 3X FLAG Peptide, SubstanceP 100 ng/mL Methionine Enkephalin, [D-Ala2]-Deltorphin II

Instrumental Analysis

Analysis of the 120 human serum samples was performed on an in-housebuilt instrument that couples a 1-m ion mobility separation (IMS-MS)with an Agilent 6224 TOF MS upgraded to a 1.5 meter flight tubeproviding resolution of 25,000 [42]. The analysis of the spiked peptidesamples and a small subset of the human serum samples was performed onboth a Thermo Fisher Scientific LTQ Orbitrap Velos MS (Velos) (San Jose,Calif., USA) operated in tandem MS (MS/MS) mode and the in-house builtIMS-MS instrument. A fully automated in-house built 4-column HPLC systemequipped with in-house packed capillary columns was used for bothinstruments with mobile phase A consisting of 0.1% formic acid in waterand phase B comprised of 0.1% formic acid in acetonitrile [43]. A100-min LC gradient was performed on the Velos MS (using 60 cm longcolumns with an o.d. of 360 μm, i.d. of 75 μm, and 3-μm C₁₈ packingmaterial), while only a 60-min gradient with shorter columns (30 cm longwith same dimensions and packing) was used with the IMS-MS. Bothgradients linearly increased mobile phase B from 0 to 60% until thefinal 2-min of the run when B was purged at 95%. 5 μL of each sample wasinjected for both analyses and the HPLC was operated under a constantflow rate of 0.4 μL/min for the 100-min gradient and 1 μL/min for the60-min gradient. The Velos MS data were collected from 400-2000 m/z at aresolution of 60,000 (automatic gain control (AGC) target: 1×10⁶)followed by data dependent ion trap MS/MS spectra (AGC target: 1×10⁴) ofthe ten most abundant ions using a collision energy setting of 35%. Adynamic exclusion time of 60 sec was used to discriminate againstpreviously analyzed ions. IMS-MS data were collected from 100-3200 m/z.

Informatics Approach and Statistical Analysis

Identification and quantification of the detected peptide peaks wereperformed utilizing the Accurate Mass and Time (AMT) tag approach(Zimmer et al., Mass Spectrom Rev, 2006. 25(3):450-82). Briefly, VelosMS/MS data were searched by SEQUEST (Thermo Scientific), and theresulting 13,448 peptides which had a MS-GF score greater than or equalto 1E-9 were used to populate an AMT tag database (Eng et al., J. Am.Soc. Mass Spec., 1994. 5(11):976-989; Kim et al., J Proteome Res, 2008.7(8):3354-63). Multiple in-house developed informatics tools (publiclyavailable ncrr.pnnl.gov/software) were used to process the LC-MS dataand correlate the resulting LC-MS features to this AMT tag database thatcontained accurate mass and LC separation elution time information forpeptide tags generated from serum proteins. Among the tools used werealgorithms for peak-picking and for determining isotopic distributionsand charge states (Jaitly et al., BMC Bioinformatics, 2009. 10(1):87).Further downstream data analysis incorporated all possible detectedpeptides into a visualization program, VIPER, to correlate LC-MSfeatures to the peptide identifications in the AMT tag database (Monroeet al., Bioinformatics, 2007. 23(15):2021-3). VIPER provided anintensity report for all detected features, normalized LC elution timesvia alignment to the database, and featured identification.

For the initial transplant data, a representative protein was chosen forredundant peptides. If a protein was identified with only onesignificant non-unique peptide it was removed. In DAnTE software,peptide peak intensity values were converted to a log 2 scalestatistically compared utilizing ANOVA (performed as a t-test with onlytwo data types in each comparison) (Polpitiya et al., Bioinformatics,2008. 24(13):1556-8). The analysis only focused on significantlychanging peptides (p-values and q-values <0.05). The q-value of a testmeasures the proportion of false positives incurred (called the falsediscovery rate) when that particular test is called significant.Significantly changing peptides were assessed at a protein level usingDAnTE's Rollup parameters (reference peptide based scaling, wherepeptides were excluded from scaling if they were not seen in at least 3datasets and no minimum peptide presence was required). Statisticallycomparisons were also done at the protein level. Only significantlychanging proteins were retained (some peptides were significant but theywere in opposing directions, so the proteins were not significant whenthe peptides were rolled up). Final significant proteins identifiedrequired at least two significant peptide identifications.

For the verification non-transplant generated data, the finalquantitative peptide identifications were independently processedthrough a series of steps including quality control, normalization,protein quantification, and comparative statistical analyses (FIG. 7).Peptide abundances were transformed to the log₁₀ scale, and fibrosisseverity categories were defined as fibrosis score=0; fibrosis score=1;and, fibrosis score=4, 5 or 6. Quality control processing was performedas previously described (Webb-Robertson et al., J Proteome Res, 2010.9(11):5748-56; Matzke et al., Bioinformatics, 2011. 27(20): 2866-72).Two main data subsets (clusters) were identified using sppPCA due tobatch differences throughout the analysis which resulted in 2 unique“subsets” within the non-transplant data (Webb-Roberston et al.,Sequential Projection Pursuit PCA—dealing with missing data associatedwith new Omics technologies. Biotechniques, 2013. in press; FIG. 8). Thetwo subsets were processed independently, starting with a quality checkto identify LC-MS runs that have significantly different peptideabundance distributions (FIGS. 9A-9D), and peptide normalization using astatistical procedure for the analysis of proteomic normalizationstrategies (SPANS) that identifies the peptide selection method and datascaling factor which introduces the least amount of bias into thedataset (Webb-Robertson et al., Proteomics, 2011. 11(24):4736-41). Foreach subset, the peptide abundance values were normalized across thetechnical replicates. Subset 1 data were normalized with mean scalingusing L order statistics (5%) peptide subset; subset 2 data werenormalized with z-score scaling using percentage of peptides present.Normalized log₁₀ abundance values were averaged across the technicalreplicates within each biological sample. For each subset, peptideabundance values were evaluated with a Tukey adjusted t-test to identifyquantitative and qualitative significance patterns, respectively.

A total of 532 proteins in subset 1 and 464 proteins in subset 2 wereestimated using a peptide signature vector approach to proteinquantitation. Peptide level significance patterns were used for proteinroll-up to select peptides within a protein that follow the samesignificance trends to estimate protein abundance. Protein abundancevalues were estimated using a standard R-rollup method as previouslydescribed (Polpitiya et al., Bioinformatics, 2008. 24(13):1556-8).Comparative statistical analyses between the 3 fibrosis severitycategories (fibrosis score=0; fibrosis score=1; fibrosis score=4, 5 or6) were performed using a Tukey adjusted t-test to assess differences inprotein average abundance.

A meta-analysis of protein significance from subsets 1 and 2 wasperformed using Fisher's Inverse Score. The meta-analysis approachprovides a mechanism that allows inference be made to proteins usingdata across many experiments. Fisher's Inverse Score approach considersthe p-values resulting from the comparative statistical analysis ofproteins. The Fisher's Inverse statistic is defined as,

S _(Fisher)=−2 ln(Π_(i)pvalue_(i))

The statistic S_(Fisher) follows a χ2 distribution with degrees offreedom (df) equal to 2 times the number of datasets. When a protein wasobserved in both experiments the df=2*2, otherwise df=2*1. Significanceis based on a p-value threshold of 0.05.

There are a combined total of 581 unique proteins and 415 in commonacross subsets 1 and 2. The results of the meta-analysis approach can besummarized in 4 parts, of which parts 1-3 are associated with proteinsthat are present in both subsets (p=415) and thus statistics can becompared between the two subsets, and part 4 shows the counts for theproteins that are unique to each subset (Table 10, Tukey adjustedt-test).

TABLE 10 Meta-analysis of protein significance using Tukey adjustedp-values. The number in meta-analysis results column (A/B) is theprotein significance after the p-values have been combined using theFishers Inverse χ2 test where A is the number significant and B is thenumber non-significant. Comparison (Score i vs. Score j) SignificanceFibrosis Score = 0 vs. Fibrosis Score = 0 vs. Fibrosis Score = 1 vs.Indicator¹ Fibrosis Score = 1 Fibrosis Score = 4/5/6 Fibrosis Score =4/5/6 Subset 1 Subset 2 Meta- Meta- Meta- (N = 42) (N = 18) Combinedanalysis Combined analysis Combined analysis Part 1. (df = 4)  1 −1 0(0/0) 0 (0/0) 0 (0/0) −1 1 0 (0/0) 0 (0/0) 0 (0/0) Part 2. (df = 4) +1+1 0 (0/0) 4 (4/0) 9 (9/0) −1 −1 3 (3/0) 15 (15/0)  17 (17/0)   0 0 304 (0/304) 184  (1/183) 187  (4/183) Part 3. (df = 4)  1 0 17 (13/4)  23(20/3)  17 (11/6)   0 1 2 (2/0) 10 (5/5) 11 (9/2) −1 0 13 (5/8) 37(23/7)  22 (18/4)   0 −1 10 (4/6) 26 (16/10) 21 (14/7)  Part 4. (df = 2)+1 NaN 2 (2/0) 3 (3/0) 2 (2/0) NaN +1 0 (0/0) 1 (1/0) 3 (3/0) −1 NaN 11(11/0)  2 (2/0) 3 (3/0) NaN −1 3 (3/0) 0 (0/0) 0 (0/0)  0 NaN 82  (0/82)68  (0/68) 66  (0/66) NaN 0 35  (0/35) 19  (0/19) 16  (0/16)¹Significance indicator value (+1) indicates the protein isstatistically significant in favor of Score j; (−1) indicates theprotein is statistically significant in favor of Score i; (0) indicatesthere is no evidence of a statistical difference between Scores i & j;and, (NaN) indicates the protein was not present in a subset.

In Part 1 of Table 10, proteins are demonstrated that have conflicting“significance” information across the two subsets. That is, thoseproteins which were found to have a statistically significant differencein favor of one score groups but in favor of the other score group forthe other subset. For the Tukey adjusted t-tests comparing proteinabundances, no proteins were found with conflicting information.

In Part 2 of Table 10, proteins are demonstrated with consistentsignificance information across the two subsets. For a small number ofproteins that are non-significant for both subsets, there is a gain insignificance after the merging due to an increase in power.

In Part 3 of Table 10, proteins are demonstrated that are significant inone subset and non-significant in the other subset. There are proteinsfor which the combination of the p-values does not give evidence ofsignificance, but others for which the combination of the significantwith the non-significant p-value again increases the power andidentifies significance.

In Part 4 of Table 10, proteins are demonstrated that are unique to onesubset or the other. A value of “NaN” means that the protein is notpresent in that subset. To combine p-values, the p-value for the missingprotein is set to 1.0 so that to achieve significance a protein wouldneed to have a p-value of less than 0.009. A balance is observed betweenthe number of proteins that are still identified as significant andthose for which the p-value increases to a non-significant level.

The meta-analysis resulted in a non-redundant candidate list of 118proteins which differentiate between 0.1 fibrosis and >4 fibrosis. Afterimplementing similar coverage requirements as the transplant sampleresults (minimum 2 significant peptides per protein), the finalcandidate list consisted of 78 proteins. To further explore theseproteins, within each subset, missing values were imputed using aregularized EM algorithm (Schneider, J. Climate, 2001. 14(5):853-871),then standardized across subsets using a z-score. A principal componentanalysis (PCA) based on the combination of the two subsets was used todetermine a linear combination of the 75 proteins that discriminatesfibrosis categories such that category 1 are those patients with a 0 or1 fibrosis score and category 2 are those patients with a 4, 5 or 6fibrosis score. The first principal component (pc) explained 26% of thetotal variation in the imputed and standardized protein abundancedataset. In addition, when the PCA scores are labeled by fibrosiscategory membership, a separation of the fibrosis categories is evident(FIG. 10).

A receiver operator characteristic (ROC) analysis was performed toassess and compare various biological metrics for their sensitivity todiscriminate the fibrosis categories. The metrics included are (1) the75 individual protein abundances (n=60), (2) the scores of the linearcombination of the 75 proteins resulting in the first pc (n=60), (3) theAPRI score (n=55), and (4) the FBS score (n=17). The scores of first pc,APRI scores and FBS scores all performed well in the discriminationproblem resulting in area under the curve (AUC) values of 1.0, 0.93 and0.98, respectively.

Western Blot Analyses

Aliquots of depleted serum samples (see above) from selected pairs offibrosis and non-fibrosis patients were used. Briefly, 5 μg of eachsample were electrophoresed using NuPage® Novex® 4-12% Bis-Tris SDS-PAGEgels (Life Technologies), alongside 5 μL SeeBlue® Plus2 Pre-stainedStandard and 5 μL MagicMark™ XP Western Protein Standard (LifeTechnologies). Proteins were transferred to PVDF membranes followed byblocking with 5% non-fat dry milk in PBS containing 0.1% Triton X-100.Membranes were incubated overnight at 4° C. with primary antibodies atdilutions of 1:1000 or 1:500. Anti-QSOX1 was used at a 1:200 dilutionfor 1 h at room temperature. Secondary antibody incubations (1:5500)were carried out for 1 h at room temperature. All antibodies were fromSanta Cruz biotechnology. SuperSignal West Femto ChemiluminescentSubstrate (Thermo Scientific) was used for detection.

Example 2 IMS-MS Developments and Improvements to Increase Sensitivityand Duty Cycle

This example describes methods that were used to improve the sensitivityof protein detection by MS.

Practical use of IMS-MS was initially impeded by its low sensitivity dueto significant ion losses at the IMS drift cell termini. This problemwas solved by re-focusing both the ions exiting the source (prior toinjection in the IMS drift cell) and those leaving the drift cell withion funnels (FIG. 1A), making the addition of the IMS stage essentiallylossless [13].

Another limitation that hindered widespread use of IMS-MS was its lowduty cycle. Traditionally ions are only pulsed into the drift cell afterall ions from the previous packet have exited, resulting in utilizationof only a small percentage of the ions created in the source. To addressthis constraint, a multiplexing approach based on the Hadamard transform[14] was developed so that discreet packets of ions could co-exist inthe drift cell as long as they did not overlap due to diffusionalbroadening (FIGS. 1B and 1C). This approach allows for much higher IMSduty cycle and has led to a significant increase in measurementsensitivity. Deconvolution of the pseudorandom sequence utilized in themultiplexing approach has also been shown to greatly reduce the noise inthe spectra allowing a much higher signal to noise ratio for theresulting ions [15]. These improvements in addition to the reducedspectral congestion from the IMS separation have enabled faster gradienttimes when combined with LC, thereby increasing the throughput of sampleanalyses [12]. To take advantage of the faster sample analyses andhigher sensitivity measurements, a LC-IMS-MS analytical platform wasdeveloped with the above sensitivity improvements for application toclinically focused large-scale proteomic measurements.

Example 3 LC-IMS-MS Platform Evaluation and Validation

This example shows application of the protein detection methods ofExample 2 to detect expression of liver fibrosis related proteins.

In an initial evaluation of the new LC-IMS-MS platform, its performancewas compared to an LC-MS platform (comprised of a commercially availableLTQ Orbitrap Velos). Nine blood serum samples were analyzed on eachplatform, and a 100-min LC gradient was used for LC-MS, while a 60-minLC gradient was used for LC-IMS-MS. Even with the shorter analysistime, >20% more deisotoped spectral features (putative peptides) weredetected with the LC-IMS-MS platform compared to the LC-MS platform(FIG. 2A), an observation attributed to the reduced spectral congestionfrom the additional IMS separation and the higher signal to noise ratiosfrom multiplexing. These attributes allow additional coverage andconfidence for the peptides observed with significant differentialabundance in the LC-IMS-MS analyses and the detection of additionalproteins not seen in the LC-MS experiments. To determine why morefeatures were observed in the LC-IMS-MS platform even with a reduced LCgradient, a follow-up limit of detection study was performed andinvolved both platforms analyzing three technical replicates of a normalhuman serum sample spiked with eight non-human peptides ranging inconcentrations from 100 pg/mL to 100 ng/mL. Overall, the LC-IMS-MSplatform detected peptides at concentrations ˜100× lower than the LC-MSplatform with a linear correlation to concentrations, lower coefficientof variation (CV) values and modestly higher throughput (60-min vs.100-min) as shown by Table 11 and FIG. 2B. These advantages illustratethe gains in enhanced dynamic range, proteome coverage, and increasedspeed for the LC-IMS-MS platform. To allow direct comparison of the TOFMS (60-min LC separation) to the LTQ Orbitrap Velos (100-minseparation), the IMS drift cell was removed. The similarities in limitsof detection for these two platforms (Table 11) showed that theincreased measurement sensitivity observed with LC-IMS-MS can beattributed specifically to the IMS separation.

TABLE 11 Scaled abundance and coefficient of variation (CV) values for 8non-human peptides spiked into human serum for 60-min LC-IMS-TOF MS,60-min LC-TOF MS and 100-min LC-LTQ Orbitrap Velos analyses PeptideScaled Abundance^(a) and CV Values^(b,c) 60-min LC-IMS- 60-min LC-100-min LC-LTQ Spiking Level Peptide TOF MS TOF MS Orbitrap Velos 100pg/mL Melittin ND ND ND 100 pg/mL Dynorphin A Porcine   1.7 (18) ND ND 1 ng/mL Des Pro Ala Bradykinin  21 (12) ND ND  1 ng/mL LeucineEnkephalin  23 (10) ND ND  10 ng/mL 3X FLAG Peptide 115 (8) 125 (20) ND 10 ng/mL Substance P 126 (7) 138 (18) 112 (19) 100 ng/mL[Ala92]-Peptide 6 868 (4) 848 (11) 841 (12) 100 ng/mL MethionineEnkephalin 1000 (3)  1000 (9)  1000 (10)  ^(a)Peptide abundance valuesfrom 3 datasets were averaged and re-scaled to a range of 0 to 1000 fordirect instrument comparison (by dividing the most abundant peptidevalue in each instrument and multiplying by 1000) ^(b)CV values are inparenthesis ^(c)ND = not detected

Example 4 Statistical Trends for Proteins with Significant DifferentialAbundance Discriminating between Fibrosis Conditions

This example shows assessment of the statistical significance of dataobtained using the protein detection methods of the preceding Examplestowards detection of liver fibrosis related proteins.

To fully evaluate its applicability, the LC-IMS-MS platform was utilizedin a study involving chronic liver disease, which is associated with avariety of origins, including viral hepatitis, alcohol abuse,nonalcoholic fatty liver disease and hepatic metabolic and immunedisorder. Specifically matched blood serum samples chosen from 60 HCVpatients following liver transplantation were initially utilized toevaluate the LC-IMS-MS platform for clinical use. These 60 samplesrepresented 30 patients termed non-progressors (NP) who showed no ormild return of fibrosis over a range of times post-liver transplant (2to 4 years), compared with 30 patients who developed “stage 3 to 4”fibrosis over a similar period of time and were stratified into eitherslow progressors (SP; stage 3-4 fibrosis at 3-4 years post-transplant)or fast progressors (FP; stage 3-4 fibrosis within 2 yearspost-transplant) (FIG. 3). The serum samples were collected at specifictime points post-transplant (ranging between 150 and 771 days with indepth information on the patient cohort available in Reference [21] andits Supporting Material). Using a multi-variant approach, the specimenswere matched for the most important clinical variables known toinfluence the risk of fibrosis progression associated with recurrent HCVafter liver transplantation: donor age and cold ischemia time of thetransplanted organ. Time to biopsy was also matched and multipleinclusion and exclusion criteria were applied, specifically patientswith known confounders for fibrosis, such as CMV infection, greater than1 episode of rejection, etc. were excluded. The specimens wereadequately found and matched for these confounders due to the extensivenumber of patient samples available through the University of WashingtonTissue Repository. Final matching resulted in 30 patient pairs(progressor vs. non-progressor), across both slow (14) and fast (16)fibrosis progression outcomes. All serum samples were analyzed utilizingthe LC-IMS-MS platform with technical replicates allowing for globalproteome evaluation of each patient sample.

Following data acquisition of the 60 HCV liver transplant serum sampleswith LC-IMS-MS, overall statistical significance was assessed, first atthe peptide level and then protein significance was evaluated aftermerging peptide with significant differential abundance valuesdistinguishing NP, SP, and FP conditions. If a protein was identifiedwith only one significant non-unique peptide it was removed. In DAnTEsoftware, peptide peak intensity values were converted to a log 2 scalestatistically compared utilizing ANOVA (performed as a t-test with onlytwo data types in each comparison) [22]. The analysis only focused onsignificantly changing peptides (p-values and q-values <0.05). Theq-value of a test measures the proportion of false positives incurred(called the false discovery rate) when that particular test is calledsignificant. Significantly changing peptides were assessed at a proteinlevel using DAnTE's Rollup parameters (reference peptide based scaling).Statistically comparisons were also done at the protein level. Onlysignificantly changing proteins were retained (some peptides weresignificant but they were in opposing directions, so the proteins werenot significant when the peptides were rolled up). Final significantproteins identified required at least two significant peptideidentifications. Statistical analysis revealed 136 differentiallyabundant proteins in the serum of transplant patients, 112 proteins wereobserved to discriminate between the NP and FP patient groups and 101proteins between NP and SP patient groups, with 77 proteins overlapping,illustrated in FIG. 4A.

Example 5 Functional Classification and Comparison with Previous Finding

This example shows contextual analysis of some of the liver fibrosisrelated proteins that show differential expression using the proteindetection methods of the preceding Examples.

Serum results were functionally classified, focusing on certainmechanisms relevant for comparison within liver function: livermetabolism, immune response (innate and adaptive), oxidative stress,architecture, and secreted effectors (Table 12, FIG. 4B). General trendsreveal a decrease in serum level markers of liver metabolism forpatients with developed fibrosis, while those related to oxidativestress increase (as seen in [23]). Mechanistically, this reflects areduction in gross metabolism, in line with attenuation of liverfunction, but appears to do so under significant oxidative stressresulting in the production of stress response proteins. As with anycomprehensive serum/plasma protein study, multiple signature immuneproteins where also captured, including alterations in adaptiveimmunoglobulin responses, acute phase and inflammatory markers, and thecomplement pathway. Though likely not specific as markers of fibrosisprogression, these signatures still reflect the gross alterationsvisible in this biofluid as a direct result of liver injury. Of specificrelevance to HCV infection however, complement C4-A exhibited lowerabundance in the SP and FP patient groups. Recent papers [24, 25] haveshown that complement C4 activity is significantly lower in HCV infectedpatients because it is transcriptionally repressed by HCV proteins,consistent with the data.

TABLE 12 Transplant Model Transplant Model Fibrosis Model FastProgressors vs their Slow Progressors vs >Stage 4 Gene matched controlstheir matched controls Fibriosis Proteins significantly increasing inthe Slow and Fast Progressors (SP & FP) compared to their matchedcontrols Oxidative Stress QSOX1 ↑ ↑ ↑ IGLC1 ↑ ↑ ↑ FCGR3A ↑ ↑ ↑ A2M ↑ ↑ ↑AFM ↑ ↑ ↑ ALCAM ↑ ↑ ↑ APOB ↑ ↑ ↓ C7 ↑ ↑ ↑ CLU ↑ ↑ ↓ Liver ArchitectureECM1 ↑ ↑ ↑ ITIH3 ↑ ↑ ↑ Liver Architecture LGALS3BP ↑ ↑ ↑ VCAM1 ↑ ↑ ↑ VWF↑ ↑ ↑ Proteins significantly decreasing in the Slow and Fast Progressors(SP & FP) compared to their matched controls Liver Metabolism F2 ↓ ↓ ↓CFI ↓ ↓ ↓ KLKB1 ↓ ↓ ↓ F11 ↓ ↓ ↓ CNDP1 ↓ ↓ ↓ Liver Metabolism BCHE ↓ ↓ ↓APCS ↓ ↓ ↓ APOH ↓ ↓ ↓ AZGP1 ↓ ↓ ↓ Immune C4A ↓ ↓ ↓ C6 ↓ ↓ ↓ C8A ↓ ↓ ↓C8B ↓ ↓ ↓ C8G ↓ ↓ ↓ HPX ↓ ↓ ↓ Liver Metabolism IGFALS ↓ ↓ ↓ ITIH1 ↓ ↓ ↓ITIH2 ↓ ↓ ↓ ITIH4 ↓ ↓ ↓ PRG4 ↓ ↓ ↓ Liver Metabolism RBP4 ↓ ↓ ↓ SERPINC1↓ ↓ ↓ SERPIND1 ↓ ↓ ↓ Liver Metabolism TTR ↓ ↓ ↓ APOC3 ↓ ↓ ↓ Proteinsshowing differentaly abundance changes in the Slow and Fast Progressors(SP & FP) compared to their matched controls - Up in FP/Down in SP LiverArchitecture F10 ↑ ↓ ↓ SERPINA4 ↑ ↓ ↓ Liver Architecture VTN ↑ ↓ ↑ LiverArchitecture LUM ↑ ↓ ↑ Proteins significantly increasing/decreasing onlyin the Slow Progressors (SP) compared to their matched controls SERPING1↑ ↑ BTD ↓ ↓ PLG ↓ ↓ PROC ↓ ↓ CPN1 ↓ ↓ CPB2 ↓ ↓ GC ↓ ↓ GP5 ↓ ↓ Proteinssignificantly increasing/decreasing in the Fast Progressors (FP)compared to their matched controls C2 ↑ ↓ Liver Architecture F12 ↑ ↓ANPEP ↑ ↑ CP ↑ ↑ Liver Architecture TGFBI ↑ ↑ FCGBP ↑ ↑ SERPINA7 ↑ ↓ PVR↑ ↑ AGT ↓ ↓ H6PD ↓ ↓ PROCR ↑ ↑ VASN ↑ ↑ Proteins significantlyincreasing/decreasing only in AK dataset Ig lambda ↑ chain V-I regionNEWM APOC1 ↑ ICAM1 ↑ CSF1R ↑ DBH ↑ COL6A3 ↑ COMP ↑ CD163 ↑ LYVE1 ↑ LCAT↓ CPN2 ↓ PROZ ↓ MASP1 ↓ SEPP1 ↓ RARRES2 ↓

Trends were observed in the panel of liver architecture proteinsallowing for differentiation of the NP, SP, and FP patient groups.Extracellular matrix proteins such as ECM1 and galectin-3-bindingproteins, which contribute to the collagenous matrix of the fibrotictissue in chronic hepatic fibrosis, both increased in SP and FP patientgroups, as well as cytoskeletal β-actin (ACTB) which increases in aradiation-induced skin and muscular fibrosis study [26]. A significantincrease was observed in FP patients for transforming growthfactor-β-induced protein ig-h3 (TGFBI) and coagulation factor XII (F12).Finally, differential responses were observed in some protein markerssuch as vitronectin (VTN), lumican (LUM), coagulation factor X (F10) andcomplement factor 5 (C5), which decrease in SP, but increase in FP.

Recent efforts report complementary mRNA and protein levelcharacterization of liver-transplant biopsy tissue collected from apatient population which overlaps, in part, with the current serumsamples [30, 31]. Previous tissue studies were longitudinal in nature,including early and late post-transplant, and accurately describe thegene and protein expression patterns and networks that differentiallydistinguish severity of fibrosis progression post-transplant. Whencomparing the liver tissue and serum results a strong oxidative stressenvironment and response directly linked to liver injury is reflected inboth liver tissue and through protein serum levels. This is confirmedthrough metabolite analysis performed on the same serum samples, showinga unique metabolic profile categorized by metabolite alterations inglutathione hemostasis and oxidative stress [30], consistent withprevious finding [32, 33]. Alterations in both adaptive and innateimmune responses are also in common and consistent with otherHCV-associated fibrosis transplant studies [30, 34]. Finally,indications of liver function attenuation (reduction in metabolism andsecretion of effectors), were also reflected in both the serum andtissue findings and correlated with fibrosis progression [30].

This initial application of the LC-IMS-MS platform demonstrates a robustcharacterization of potential differentiating serum proteins, withfaster throughput (40% gain) while maintaining or exceeding sensitivitycompared to current conventional platforms (Table 11).

Example 6 Non-Transplant Patient Serum Verification Analysis andComparison

This example shows validation of results obtained on transplant patientswith liver fibrosis using protein detection methods of the precedingExamples against results obtained using the methods on non-transplantliver fibrosis subjects.

Investigations utilizing a liver transplant patient population confermultiple advantages in studies involving fibrosis progression (part ofwhich is the rapidity with which fibrosis develops post-transplant), butit is recognized that such studies also introduce clinical confoundersand variables not mechanistically linked to progression of liverfibrosis but directly related to the transplantation itself andimmunosuppressive environments. These factors likely add analyticalcomplexity to an already dynamic clinical investigation. To provideclarity towards significant findings from the transplant model and atthe same time verify the subset of detected serum proteins which aredirectly related to fibrosis progression, the LC-IMS-MS platformdescribed above was utilized to survey a completely independentHCV-infected but non-transplant patient cohort). A total of 60 patientserum samples were identified and analyzed based upon fibrosisstratification of Ishak score 0-1 versus 4-6, with timeframe of samplingwithin 6 months of a diagnostic biopsy as shown in FIG. 5A.

Statistical analysis of the subsequent peptide and protein results wasperformed using an independent analysis pipeline (See Example 1 fordetails) which resulted in the orthogonal identification of 78 proteins(see Table 5) which differentiate between the fibrosis classes (FIG.5B). Notably, 81% of these proteins (63) overlap with those thatsignificantly differentiate fibrosis progression in the initialtransplant patient results, with the majority (43) overlapping withthose in common between SP and FP. Additionally, after excluding 4proteins which decrease in SP, but increase in FP, >91% are observedwith common abundance directionality, providing a strong orthogonalvalidation of core proteins based upon both transplant andnon-transplant studies. Furthermore, of the 14 proteins uniquelysignificant in the non-transplant data, half have supporting significantpeptides in the transplant data, but were initially excluded due to lackof multiple peptides per protein. (See Table 12 for protein overlapinformation)

Beyond validation purposes, comparison of the transplant andnon-transplant results provides evidence concerning the liver fibrosisspecificity of serum protein levels, and aids in identifying signatureswhich may be more driven by the transplant environment. The coreobservations previously observed with the transplant study still hold inthe non-transplant results. Specifically, as seen in the proteinsdepicted in FIG. 4B, decreases are still consistent in liver metabolism,proteins F2, BCHE, RBP4, TTHY and IGFALS; decreasing immune responseprotein CO4A; increasing oxidative stress protein QSOX1; and increasingliver architecture proteins ECM1, LGALS3BP and TGFBI. Other verifiedproteins previously described include HPX, AGT, PROC and A2M. Thus, insome examples the disclosed methods and systems use at least (a) F2,BCHE, RBP4, TTHY, IGFALS, CO4A, QSOX1, ECM1, LGALS3BP and TGFBI, or (b)F2, BCHE, RBP4, TTHY, IGFALS, CO4A, QSOX1, ECM1, LGALS3BP, TGFBI, HPX,AGT, PROC and A2M. With regard to the subset of serum markers found onlywith transplant results, a much more robust adaptive immune response isseen, in both the number and magnitude of immunoglobulin related serumidentifications, which is partially observed in the non-transplantresults (Table 12). Additionally, a number of general hepatic classicalserum/plasma proteins, ALB, ORM2, ORM1, A1AT, HBB, HP, APOA1, APOA2 andCO3, were also uniquely significant in the transplant model but notverified in the non-transplant results. Interestingly most of theseproteins were immunoaffinity depleted prior to platform analysis, sotheir initial differential abundance was based upon either detection ofan altered protein state (confirmation change, denature or degradedform), or their protein abundance was altered enough to overwhelmcapture on the affinity column.

Example 7 Comparison with Current Metrics of Fibrosis Progression

This example compares fibrosis tracking performance obtainable using theLC-IMS-MS improved sensitivity protein detection method against currentmetrics for fibrosis progression. This example also validates resultsfrom the LC-IMS-MS platform for five proteins showing differentialexpression in both the transplant and non-transplant patient groupsusing Western blot immunoassays.

There are several current clinically applied tests and measures to trackfibrosis progression in patients, two of which, APRI calculation [39]and FIBROSpect score [40], where measured herein and were compared withthe identified protein signatures. Using the non-transplant results, areceiver operator characteristic (ROC) comparative analysis wasperformed comparing 75 identified and quantified protein values (78minus the removal of immunoglobulin IDs) with measured FIBROSpect scores(n=17) and the calculated APRI score (n=60).

The set of 75 serum proteins detected by LC-IMS-MS was able to achieve afull discrimination of patients, area under the curve (AUC)=1, comparedto FIBROSpect AUC=0.98 and APRI AUC=0.93. FIBROSpect results are in linewith previous studies which showed its clear utility to discriminationbetween advanced and mild disease (0.1 Ishak versus >4 Ishak) [41].However there are limitations for both sensitivity and selectivity whenreporting the FIBROSpect middle test range (correlation to Ishak 2-3)[41]. The current protein markers performed as well as FIBROSpect scoresin discrimination of mild versus advanced disease, and a comprehensivelist of markers, as presented, may perform well in middle diseasedetermination, which would be a significant benefit over currentlyavailable measures.

To provide a more traditional validation of the results from theLC-IMS-MS platform, a limited subset of Western blot immunoassays wereperformed on five proteins with significant differential abundance inboth the transplant and non-transplant patients groups (F2, C4A, QSOX1,ECM1 and LGALS3BP). Within the LC-IMS-MS studies, F2 and C4A bothdecrease in patients with fibrosis while QSOX1, ECM1 and LGALS3BPincrease. These results were essentially mirrored in the immunoassayblot results where two transplant NP-FP patient pair serum samples wereblotted for each protein as shown in FIG. 6. The corresponding bargraphs represent the LC-IMS-MS measured protein values, providing anorthogonal validation of LC-IMS-MS platform.

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In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the invention. Rather, the scope of thedisclosure is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

We claim:
 1. A method of diagnosing or prognosing liver fibrosis in asubject comprising: detecting at least two, at least 3, or at least 4liver fibrosis-related molecules listed in any of Tables 1, 2, 3, 5, 6and 12 and any of SEQ ID NOS: 1-5544 in a sample obtained from thesubject; comparing expression of the at least two liver fibrosis-relatedmolecules to controls representing expression of the at least two liverfibrosis-related molecules expected in a subject who does not have liverfibrosis; and diagnosing or prognosing liver fibrosis in the subjectwhen differential expression of the at least two liver fibrosis-relatedmolecules between the sample and the control is detected, whereindifferential expression comprises an increase or decrease of at least10% relative to the controls.
 2. The method of claim 1, whereindiagnosing or prognosing liver fibrosis comprises diagnosing orprognosing fast-progressing liver fibrosis or slow-progressing liverfibrosis.
 3. The method of claim 1, wherein the at least two liverfibrosis-related molecules are listed in: Table 2; Table 3 or Table 5.4. The method of claim 1, wherein expression of at least, five, six,seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen,sixteen, seventeen, eighteen or nineteen liver fibrosis-relatedmolecules are detected.
 5. The method of claim 1, wherein the subject isdiagnosed or prognosed with liver fibrosis when differential expressionof the at least two liver fibrosis-related molecules between the sampleand the control is detected, wherein differential expression is detectedfor at least 80% of the at least two liver fibrosis-related molecules.6. The method of claim 1, wherein the method is a method of diagnosingor prognosing fast-progressing liver fibrosis, and wherein the methodcomprises: diagnosing or prognosing fast-progressing liver fibrosis whendifferential expression of the at least two liver fibrosis-relatedmolecules between the sample and the control is detected, wherein the atleast two liver fibrosis-related molecules are selected from thoselisted in Section B of Table 6; or diagnosing or prognosingfast-progressing liver fibrosis when increased expression of the atleast two liver fibrosis-related molecules in the sample is detected,wherein the at least two liver fibrosis-related molecules are selectedfrom the group consisting of F10, FN1, C5, SERPINA4, VTM and LUM.
 7. Themethod of claim 1, wherein the method is a method of diagnosing orprognosing slow-progressing liver fibrosis, and wherein the methodcomprises: diagnosing or prognosing slow-progressing liver fibrosis whendifferential expression of the at least two liver fibrosis-relatedmolecules between the sample and the control is detected, wherein the atleast two liver fibrosis-related molecules are selected from thoselisted in Section A of Table 6, or diagnosing or prognosingslow-progressing liver fibrosis when decreased expression of the atleast two liver fibrosis-related molecules between in the blood sampleis detected, wherein the at least two protein liver fibrosis-relatedmolecules are selected from the group consisting of F10, FN1, C5,SERPINA4, VTM and LUM.
 8. The method of claim 1, wherein the controlsare reference values for a population of subjects who do not have liverfibrosis or who has non-progressing liver fibrosis.
 9. The method ofclaim 1, wherein the subject has or is suspected of havingpost-transplant liver fibrosis and method is performed at least 30 daysafter liver transplant.
 10. The method of claim 1, wherein the method:has a sensitivity of at least 95% and a specificity of at least 95% forthe detection of liver fibrosis; has a sensitivity of at least 95% and aspecificity of at least 95% for identifying fast-progressing liverfibrosis; or combinations thereof.
 11. The method of claim 1, whereindifferential expression of the at least two liver fibrosis-relatedmolecules is detected within 30 days after the onset of pathologicalfibrotic change in the liver.
 12. The method of claim 1, whereindifferential expression of the at least two liver fibrosis-relatedmolecules is detected at least 180 days before onset of clinical signsor symptoms that indicate liver fibrosis or at least 90 days beforeelevation in aspartate aminotransferase (AST), alanine aminotransferase(ALT) or alkaline phosphatase (AP).
 13. The method of claim 1, wherein:differential expression of the at least two liver fibrosis-relatedmolecules is detected at least 100 days before onset of clinical signsor symptoms that indicate fast-progressing liver fibrosis; ordifferential expression of the at least two liver fibrosis-relatedmolecules is detected at least 100 days before onset of clinical signsor symptoms that indicate slow-progressing liver fibrosis.
 14. Themethod of claim 1, wherein the method further comprises detectingexpression of one or more control proteins or nucleic acid molecules.15. The method of claim 1, further comprising treating the fibrosis whenthe subject is diagnosed or prognosed with liver fibrosis.
 16. Themethod of claim 1, wherein detecting the at least two liverfibrosis-related molecules comprises analyzing the sample using ionmobility spectrometry and mass spectrometry.
 17. The method of claim 1,wherein detecting at least two liver fibrosis-related moleculescomprises quantification of the at least two liver fibrosis-relatedmolecules.
 18. A kit for the diagnosis or prognosis of liver fibrosis ina subject, comprising: reagents for detecting at least two liverfibrosis-related molecules listed in any of Tables 1, 2, 3, 5, 6 and 12or any of SEQ ID NOS: 1-5544, each in a separate container.
 19. The kitof claim 18, wherein the reagents comprise antibodies specific for theat least two liver fibrosis-related molecules listed in any of Tables 1,2, 3, 5, 6 and 12 or any of SEQ ID NOS: 1-5544.
 20. The kit of claim 18,wherein the kit further comprises: labeled secondary antibodies specificfor the antibodies specific for the at least two liver fibrosis-relatedmolecules listed in any of Tables 1, 2, 3, 5, 6 and 12 or any of SEQ IDNOS: 1-5544; a substrate, to which the antibodies specific for the atleast two liver fibrosis-related molecules listed in any of Tables 1, 2,3, 5, 6 and 12 or as any of SEQ ID NOS: 1-5544, is attached; an alarmwhich indicates the presence of liver fibrosis, wherein the alarm isactivated if at least two liver fibrosis-related molecules are detectedwith differential expression relative to control levels for a subjectwithout liver fibrosis, wherein differential expression comprises anincrease or decrease of at least 10%; an alarm which indicates thepresence of liver fibrosis, wherein the alarm is activated if at least80% of the at least two liver-fibrosis molecules are detected withdifferential expression relative to control levels for a subject withoutliver fibrosis or with non-progressing liver fibrosis, whereindifferential expression comprises an increase or decrease of at least10%; an interface configured to accept input of a criterion fordiagnosis or prognosis of liver fibrosis; an interface configured toaccept input of a selection of at least two liver fibrosis-relatedmolecules; or combinations thereof.
 21. The kit of claim 18, wherein thekit comprises reagents for detecting at least two of the liverfibrosis-related molecules listed in: Table 2, Table 3, Table 5, any ofSEQ ID NOS: 1-5544; the group consisting of QSOX1, ECM1, LGALS3BP,lumican, vitronectin, DOPA (DBH), Transthyretin (TTR), cholinesterase(BCHE), retinol-binding protein (RBP4), and IGFALS.